Compositions comprising enzyme-cleavable oxycodone prodrug

ABSTRACT

The embodiments provide Compound KC-8, N-1-[3-(oxycodone-6-enol-carbonyl-methyl-amino)-2,2-dimethyl-propylamine]-arginine-glycine-malonic acid, or acceptable salts, solvates, and hydrates thereof. The present disclosure also provides compositions, and their methods of use, where the compositions comprise a prodrug, Compound KC-8, that provides controlled release of oxycodone. Such compositions can optionally provide a trypsin inhibitor that interacts with the enzyme that mediates the controlled release of oxycodone from the prodrug so as to attenuate enzymatic cleavage of the prodrug.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/346,062, filed Jan. 9, 2012, now U.S. Pat. No. 8,569,228 which claimsthe benefit of U.S. Provisional Application No. 61/431,781 filed Jan.11, 2011.

Ketone-containing opioids, such as hydrocodone and oxycodone, aresusceptible to misuse, abuse, or overdose. Use of and access to thesedrugs therefore needs to be controlled. The control of access to thedrugs is expensive to administer and can result in denial of treatmentfor patients that are not able to present themselves for dosing. Forexample, patients suffering from acute pain may be denied treatment withan opioid unless they have been admitted to a hospital. Furthermore,control of use is often ineffective, leading to substantial morbidityand deleterious social consequences.

SUMMARY

The embodiments provide Compound KC-8,N-1-[3-(oxycodone-6-enol-carbonyl-methyl-amino)-2,2-dimethyl-propylamine]-arginine-glycine-malonicacid, shown below:

or acceptable salts, solvates, and hydrates thereof. Compound KC-8 is aprodrug that provides controlled release of oxycodone.

The embodiments provide a composition, which comprisesN-1-[3-(oxycodone-6-enol-carbonyl-methyl-amino)-2,2-dimethyl-propylamine]-arginine-glycine-malonicacid, Compound KC-8, shown below:

or pharmaceutically acceptable salts, solvates, and hydrates thereof.

The disclosure provides Compound KC-8, a ketone-modified opioid prodrugthat provides controlled release of oxycodone. In a ketone-modifiedopioid prodrug, a promoiety is attached to oxycodone through the enolicoxygen atom of oxycodone. In a ketone-modified opioid prodrug, thehydrogen atom of the corresponding enolic group of oxycodone is replacedby a covalent bond to a promoiety. The promoiety comprises anenzyme-cleavable moiety and a cyclizable spacer leaving group such thatCompound KC-8 provides controlled release of oxycodone via enzymecleavage followed by intramolecular cyclization. Compound KC-8 providesefficient delivery of oxycodone when ingested.

The present disclosure also provides pharmaceutical compositions, andtheir methods of use, where the pharmaceutical compositions comprise aprodrug, Compound KC-8, that provides controlled release of oxycodonevia enzyme cleavage followed by intramolecular cyclization. Suchcompositions can optionally provide an inhibitor, such as a trypsininhibitor, that interacts with the enzyme that mediates the controlledrelease of oxycodone from the prodrug so as to attenuate enzymaticcleavage of the prodrug. The disclosure provides for the enzyme being agastrointestinal (GI) enzyme, such as trypsin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representing the effect of increasing the level ofa trypsin inhibitor (“inhibitor”, X axis) on a PK parameter (e.g., drugCmax) (Y axis) for a fixed dose of prodrug. The effect of inhibitor upona prodrug PK parameter can range from undetectable, to moderate, tocomplete inhibition (i.e., no detectable drug release).

FIG. 2 provides schematics of drug concentration in plasma (Y axis) overtime. Panel A is a schematic of a pharmacokinetic (PK) profile followingingestion of prodrug with a trypsin inhibitor (dashed line) where thedrug Cmax is modified relative to that of prodrug without inhibitor(solid line). Panel B is a schematic of a PK profile following ingestionof prodrug with inhibitor (dashed line) where drug Cmax and drug Tmaxare modified relative to that of prodrug without inhibitor (solid line).Panel C is a schematic of a PK profile following ingestion of prodrugwith inhibitor (dashed line) where drug Tmax is modified relative tothat of prodrug without inhibitor (solid line).

FIG. 3 provides schematics representing differential concentration-dosePK profiles that can result from the dosing of multiples of a dose unit(X axis) of the present disclosure. Different PK profiles (asexemplified herein for a representative PK parameter, drug Cmax (Yaxis)) can be provided by adjusting the relative amount of prodrug andtrypsin inhibitor contained in a single dose unit or by using adifferent prodrug or inhibitor in the dose unit.

FIG. 4 compares mean plasma concentrations over time of oxycodonerelease following PO administration to rats of increasing doses ofprodrug Compound KC-8.

FIG. 5 compares mean plasma concentrations over time of oxycodonefollowing PO administration to dogs of prodrug Compound KC-8, prodrugCompound KC-3, OxyContin® tablets, or oxycodone HCl.

FIG. 6A and FIG. 6B compare mean plasma concentrations over time ofoxycodone release following PO administration to rats of two doses ofCompound KC-8, each co-dosed with increasing amounts of trypsininhibitor Compound 109.

FIG. 7A compares mean plasma concentrations over time of oxycodonerelease following PO administration to rats of single and multiple dosesof prodrug Compound KC-8 in the absence of trypsin inhibitor. FIG. 7Bcompares mean plasma concentrations over time of oxycodone releasefollowing PO administration to rats of single and multiple dose unitscomprising prodrug Compound KC-8 and trypsin inhibitor Compound 109.

FIG. 8 compares mean plasma concentrations over time of oxycodonerelease following PO administration to dogs of prodrug Compound KC-8 inthe absence or presence of trypsin inhibitor Compound 109.

TERMS

The following terms have the following meaning unless otherwiseindicated. Any undefined terms have their art recognized meanings.

“Dose unit” as used herein refers to a combination of atrypsin-cleavable prodrug (e.g., trypsin-cleavable prodrug) and atrypsin inhibitor. A “single dose unit” is a single unit of acombination of a trypsin-cleavable prodrug (e.g., trypsin-cleavableprodrug) and a trypsin inhibitor, where the single dose unit provide atherapeutically effective amount of drug (i.e., a sufficient amount ofdrug to effect a therapeutic effect, e.g., a dose within the respectivedrug's therapeutic window, or therapeutic range). “Multiple dose units”or “multiples of a dose unit” or a “multiple of a dose unit” refers toat least two single dose units.

“Gastrointestinal enzyme” or “GI enzyme” refers to an enzyme located inthe gastrointestinal (GI) tract, which encompasses the anatomical sitesfrom mouth to anus. Trypsin is an example of a GI enzyme.

“Gastrointestinal enzyme-cleavable moiety” or “GI enzyme-cleavablemoiety” refers to a group comprising a site susceptible to cleavage by aGI enzyme. For example, a “trypsin-cleavable moiety” refers to a groupcomprising a site susceptible to cleavage by trypsin.

“Gastrointestinal enzyme inhibitor” or “GI enzyme inhibitor” refers toany agent capable of inhibiting the action of a gastrointestinal enzymeon a substrate. The term also encompasses salts of gastrointestinalenzyme inhibitors. For example, a “trypsin inhibitor” refers to anyagent capable of inhibiting the action of trypsin on a substrate.

“Patient” includes humans, and also other mammals, such as livestock,zoo animals and companion animals, such as a cat, dog or horse.

“Pharmaceutical composition” refers to at least one compound and canfurther comprise a pharmaceutically acceptable carrier, with which thecompound is administered to a patient.

“Pharmaceutically acceptable carrier” refers to a diluent, adjuvant,excipient or vehicle with, or in which a compound is administered.

“Pharmaceutically acceptable salt” refers to a salt of a compound, whichpossesses the desired pharmacological activity of the compound. Suchsalts include: (1) acid addition salts, formed with inorganic acids suchas hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or formed with organic acids such asacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid,glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the compound is replacedby a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or analuminum ion; or coordinates with an organic base such as ethanolamine,diethanolamine, triethanolamine, N-methylglucamine and the like.

“Pharmacodynamic (PD) profile” refers to a profile of the efficacy of adrug in a patient (or subject or user), which is characterized by PDparameters. “PD parameters” include “drug Emax” (the maximum drugefficacy), “drug EC50” (the concentration of drug at 50% of the Emax)and side effects.

“PK parameter” refers to a measure of drug concentration in blood orplasma, such as: 1) “drug Cmax”, the maximum concentration of drugachieved in blood or plasma; 2) “drug T_(max)”, the time elapsedfollowing ingestion to achieve Cmax; and 3) “drug exposure”, the totalconcentration of drug present in blood or plasma over a selected periodof time, which can be measured using the area under the curve (AUC) of atime course of drug release over a selected period of time (t).Modification of one or more PK parameters provides for a modified PKprofile.

“PK profile” refers to a profile of drug concentration in blood orplasma. Such a profile can be a relationship of drug concentration overtime (i.e., a “concentration-time PK profile”) or a relationship of drugconcentration versus number of doses ingested (i.e., a“concentration-dose PK profile”). A PK profile is characterized by PKparameters.

“Preventing” or “prevention” or “prophylaxis” refers to a reduction inrisk of occurrence of a condition, such as pain.

“Prodrug” refers to a derivative of an active agent that requires atransformation within the body to release the active agent. In certainembodiments, the transformation is an enzymatic transformation. Incertain embodiments, the transformation is a cyclization transformation.In certain embodiments, the transformation is a combination of anenzymatic transformation and a cyclization reaction. Prodrugs arefrequently, although not necessarily, pharmacologically inactive untilconverted to the active agent.

“Promoiety” refers to a form of protecting group that, when used to maska functional group within an active agent, converts the active agentinto a prodrug. Typically, the promoiety will be attached to the drugvia bond(s) that are cleaved by enzymatic or non-enzymatic means invivo.

“Solvate” as used herein refers to a complex or aggregate formed by oneor more molecules of a solute, e.g. a prodrug or apharmaceutically-acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent. Representativesolvents include by way of example, water, methanol, ethanol,isopropanol, acetic acid, and the like. When the solvent is water, thesolvate formed is a hydrate.

“Therapeutically effective amount” means the amount of a compound (e.g.,prodrug) that, when administered to a patient for preventing or treatinga condition such as pain, is sufficient to effect such treatment. The“therapeutically effective amount” will vary depending on the compound,the condition and its severity and the age, weight, etc., of thepatient.

“Treating” or “treatment” of any condition, such as pain, refers, incertain embodiments, to ameliorating the condition (i.e., arresting orreducing the development of the condition). In certain embodiments“treating” or “treatment” refers to ameliorating at least one physicalparameter, which may not be discernible by the patient. In certainembodiments, “treating” or “treatment” refers to inhibiting thecondition, either physically, (e.g., stabilization of a discerniblesymptom), physiologically, (e.g., stabilization of a physicalparameter), or both. In certain embodiments, “treating” or “treatment”refers to delaying the onset of the condition.

DETAILED DESCRIPTION

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It should be understood that as used herein, the term “a” entity or “an”entity refers to one or more of that entity. For example, a compoundrefers to one or more compounds. As such, the terms “a”, “an”, “one ormore” and “at least one” can be used interchangeably. Similarly theterms “comprising”, “including” and “having” can be usedinterchangeably.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

Except as otherwise noted, the methods and techniques of the presentembodiments are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification. See, e.g., Loudon, Organic Chemistry, Fourth Edition, NewYork: Oxford University Press, 2002, pp. 360-361, 1084-1085; Smith andMarch, March's Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, Fifth Edition, Wiley-Interscience, 2001.

The nomenclature used herein to name the subject compounds isillustrated in the Examples herein. In certain instances, thisnomenclature is derived using the commercially-available AutoNomsoftware (MDL, San Leandro, Calif.).

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the chemical groups represented by the variables arespecifically embraced by the present invention and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed, to the extent that such combinations embrace compounds thatare stable compounds (i.e., compounds that can be isolated,characterised, and tested for biological activity). In addition, allsub-combinations of the chemical groups listed in the embodimentsdescribing such variables are also specifically embraced by the presentinvention and are disclosed herein just as if each and every suchsub-combination of chemical groups was individually and explicitlydisclosed herein.

General Synthetic Procedures

Many general references providing commonly known chemical syntheticschemes and conditions useful for synthesizing the disclosed compoundsare available (see, e.g., Smith and March, March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, Fifth Edition,Wiley-Interscience, 2001; or Vogel, A Textbook of Practical OrganicChemistry, Including Qualitative Organic Analysis, Fourth Edition, NewYork: Longman, 1978).

Compounds as described herein can be purified by any of the means knownin the art, including chromatographic means, such as high performanceliquid chromatography (HPLC), preparative thin layer chromatography,flash column chromatography and ion exchange chromatography. Anysuitable stationary phase can be used, including normal and reversedphases as well as ionic resins. See, e.g., Introduction to Modern LiquidChromatography, 2nd Edition, ed. L. R. Snyder and J. J. Kirkland, JohnWiley and Sons, 1979; and Thin Layer Chromatography, ed E. Stahl,Springer-Verlag, New York, 1969.

During any of the processes for preparation of the compounds of thepresent disclosure, it may be necessary and/or desirable to protectsensitive or reactive groups on any of the molecules concerned. This canbe achieved by means of conventional protecting groups as described instandard works, such as T. W. Greene and P. G. M. Wuts, “ProtectiveGroups in Organic Synthesis”, Fourth edition, Wiley, New York 2006. Theprotecting groups can be removed at a convenient subsequent stage usingmethods known from the art.

The compounds described herein can contain one or more chiral centersand/or double bonds and therefore, can exist as stereoisomers, such asdouble-bond isomers (i.e., geometric isomers), enantiomers ordiastereomers. Accordingly, all possible enantiomers and stereoisomersof the compounds including the stereoisomerically pure form (e.g.,geometrically pure, enantiomerically pure or diastereomerically pure)and enantiomeric and stereoisomeric mixtures are included in thedescription of the compounds herein. Enantiomeric and stereoisomericmixtures can be resolved into their component enantiomers orstereoisomers using separation techniques or chiral synthesis techniqueswell known to the skilled artisan. The compounds can also exist inseveral tautomeric forms including the enol form, the keto form andmixtures thereof. Accordingly, the chemical structures depicted hereinencompass all possible tautomeric forms of the illustrated compounds.

The compounds described also include isotopically labeled compoundswhere one or more atoms have an atomic mass different from the atomicmass conventionally found in nature. Examples of isotopes that can beincorporated into the compounds disclosed herein include, but are notlimited to, ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, etc. Compounds canexist in unsolvated forms as well as solvated forms, including hydratedforms. In general, compounds can be hydrated or solvated. Certaincompounds can exist in multiple crystalline or amorphous forms. Ingeneral, all physical forms are equivalent for the uses contemplatedherein and are intended to be within the scope of the presentdisclosure.

Representative Embodiments

Reference will now be made in detail to various embodiments. It will beunderstood that the invention is not limited to these embodiments. Tothe contrary, it is intended to cover alternatives, modifications, andequivalents as may be included within the spirit and scope of theallowed claims.

The embodiments provide Compound KC-8,N-1-[3-(oxycodone-6-enol-carbonyl-methyl-amino)-2,2-dimethyl-propylamine]-arginine-glycine-malonicacid, shown below:

or acceptable salts, solvates, and hydrates thereof.

The embodiments provide a composition, which comprisesN-1-[3-(oxycodone-6-enol-carbonyl-methyl-amino)-2,2-dimethyl-propylamine]-arginine-glycine-malonicacid, Compound KC-8, shown below:

or pharmaceutically acceptable salts, solvates, and hydrates thereof.

The disclosure provides Compound KC-8, a ketone-modified opioid prodrugthat provides controlled release of oxycodone. In a ketone-modifiedopioid prodrug, a promoiety is attached to oxycodone through the enolicoxygen atom of oxycodone. In a ketone-modified opioid prodrug, thehydrogen atom of the corresponding enolic group of oxycodone is replacedby a covalent bond to a promoiety.

In Compound KC-8, the promoiety comprises a cyclizable spacer leavinggroup and a cleavable moiety. In Compound KC-8, the ketone-modifiedoxycodone prodrug is a corresponding compound in which the enolic oxygenatom has been substituted with a spacer leaving group bearing a nitrogennucleophile that is protected with an enzyme-cleavable moiety, theconfiguration of the spacer leaving group and nitrogen nucleophile beingsuch that, upon enzymatic cleavage of the cleavable moiety, the nitrogennucleophile is capable of forming a cyclic urea, liberating the compoundfrom the spacer leaving group so as to provide oxycodone.

The enzyme capable of cleaving the enzyme-cleavable moiety may be apeptidase, also referred to as a protease—the promoiety comprising theenzyme-cleavable moiety being linked to the nucleophilic nitrogenthrough an amide (e.g. a peptide: —NHC(O)—) bond. In some embodiments,the enzyme is a digestive enzyme of a protein. The disclosure providesfor the enzyme being a GI enzyme, such as trypsin and for theenzyme-cleavable moiety being a GI enzyme-cleavable moiety, such as atrypsin-cleavable moiety.

The corresponding prodrug provides post administration-activated,controlled release of oxycodone. The prodrug requires enzymatic cleavageto initiate release of oxycodone, and thus the rate of release ofoxycodone depends upon both the rate of enzymatic cleavage and the rateof cyclization. Compound KC-8 provides efficient controlled release ofoxycodone due to a combination of a rapid enzyme cleavage rate and arapid intramolecular cyclization rate. The prodrug is configured so thatit will not provide excessively high plasma levels of the active drug ifit is administered inappropriately, and cannot readily be decomposed toafford the active drug other than by enzymatic cleavage followed bycontrolled cyclization.

The cyclic group formed when oxycodone is released is convenientlypharmaceutically acceptable, in particular a pharmaceutically acceptablecyclic urea. It will be appreciated that cyclic ureas are generally verystable and have low toxicity.

General Synthetic Procedures for Compounds

Representative synthetic schemes for compounds disclosed herein areshown below. Compound KC-8 can be synthesized by using the disclosedmethods.

Representative Synthetic Schemes

A representative synthesis for Compound S-104 is shown in Scheme 1. InScheme 1, for Compound KC-8, n is 3; the first and third geminal R¹ andR² are hydrogen; the second geminal R¹ and R² are methyl; R⁵ is methyl;and PG¹ is an amino protecting group.

In Scheme 1, Compound 5-100 is a commercially available startingmaterial. Alternatively, Compound S-100 can be synthesized via a varietyof different synthetic routes using commercially available startingmaterials and/or starting materials prepared by conventional syntheticmethods.

With continued reference to Scheme 1, Compound S-100 is protected at theamino group with a trifluoroacetyl group to form Compound S-101. Atrifluoroacetyl group can be formed with reaction using reagents, suchas ethyl trifluoroacetate, trifluoroacetyl chloride, or1,1,1-trichloro-3,3,3-trifluoroacetone.

Compound S-101 is protected at the other amino group to form CompoundS-102, wherein PG¹ is an amino protecting group. Amino protecting groupscan be found in T. W Greene and P. G. M. Wuts, “Protective Groups inOrganic Synthesis”, Fourth edition, Wiley, New York 2006. Representativeamino-protecting groups include, but are not limited to, formyl groups;acyl groups, for example alkanoyl groups, such as acetyl; alkoxycarbonylgroups, such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl groups,such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc);arylmethyl groups, such as benzyl (Bn), trityl (Tr), and1,1-di-(4′-methoxyphenyl)methyl; silyl groups, such as trimethylsilyl(TMS) and tert-butyldimethylsilyl (TBS); and the like.

In certain embodiments, PG¹ is Boc. Conditions for forming Boc groups onCompound S-102 can be found in Greene and Wuts. One method is reactionof Compound S-101 with di-tert-butyl dicarbonate. The reaction canoptionally be run in the presence of an activating agent, such as DMAP.In Scheme 1, in certain embodiments, the trifluoroacetyl protectinggroup and PG¹, such as Boc, are orthogonal protecting groups.

With continued reference to Scheme 1, the trifluoroacetyl group onCompound S-102 is deprotected to form Compound S-103. Conditions toremove the trifluoroacetyl group can be found in Greene and Wuts.Methods to remove the trifluoroacetyl group include hydrolysis ofCompound S-102. Certain conditions for hydrolysis include reaction withsodium hydroxide or lithium hydroxide.

With continued reference to Scheme 1, the R⁵ group is added ontoCompound S-103 to form Compound S-104. The addition of the R⁵ group ontothe amino group of Compound S-103 can be facilitated with the use ofprotecting/activating groups. In certain embodiments, a nosyl group isadded onto the amino group of Compound S-103 before addition of the R⁵group. A nosyl group can be added with use of nosyl chloride.

In certain embodiments, the R⁵ group is methyl and is added through useof methyl iodide. After addition of the R⁵ group, theprotecting/activating group can be removed to yield Compound S-104. Forexample, removal of the nosyl group can be performed with thiophenol.

A representative synthesis for Compound S-202 is shown in Scheme 2. InScheme 2, for Compound KC-8, R^(a) is hydroxyl; n is 3; the first andthird geminal R¹ and R² are hydrogen; the second geminal R¹ and R² aremethyl; R⁵ is methyl; and PG¹ is an amino protecting group.

In Scheme 2, Compound S-200 is a commercially available startingmaterial. Alternatively, Compound S-200 can be semi-syntheticallyderived from natural materials or synthesized via a variety of differentsynthetic routes using commercially available starting materials and/orstarting materials prepared by conventional synthetic methods.

With continued reference to Scheme 2, Compound S-200 is reacted withCompound S-104 to form Compound S-201. In this reaction, Compound S-200reacts with the amino group of Compound S-104 with a carbamate-formingreagent to yield Compound S-201. Suitable carbamate-forming reagentsinclude chloroformates, such as 4-nitrophenyl chloroformate.

With continued reference to Scheme 2, the protecting group PG¹ isremoved from Compound S-201 to form Compound S-202. Conditions to removeamino groups can be found in Greene and Wuts. When PG¹ is a Boc group,the protecting group can be removed with acidic conditions, such astreatment with hydrochloric acid or trifluoroacetic acid.

A representative synthesis for Compound S-303 is shown in Scheme 3. InScheme 3, for Compound KC-8, R^(a) is hydroxyl; n is 3; the first andthird geminal R¹ and R² are hydrogen; the second geminal R¹ and R² aremethyl; R⁵ is methyl; R⁶ is the side chain of arginine; and PG² is anoptional amino protecting group.

With reference to Scheme 3, Compound S-202 reacts with Compound S-301 toform Compound S-302 in a peptide coupling reaction. In certainembodiments, R⁶ is the side chain of arginine and is optionallyprotected. Protecting groups for the side chain of arginine are known tothose skilled in art and can be found in Greene and Wuts. In certaininstances, the protecting group for the side chain of arginine is asulfonyl-type protecting group, such as2,2,4,6,7-pentamethyldihydrobenzofurane (Pbf). Other protecting groupsinclude 2,2,5,7,8-pentamethylchroman (Pmc) and1,2-dimethylindole-3-sulfonyl (MIS).

A peptide coupling reaction typically employs a conventional peptidecoupling reagent and is conducted under conventional coupling reactionconditions, typically in the presence of a trialkylamine, such asethyldiisopropylamine or diisopropylethylamine (DIEA). Suitable couplingreagents for use include, by way of example, carbodiimides, such asethyl-3-(3-dimethylamino)propylcarbodiimide (EDC),dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) and thelike, and other well-known coupling reagents, such asN,N′-carbonyldiimidazole, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline(EEDQ), benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP),O-(7-azabenzotriazol-1-yl)-N,N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) and the like. Optionally, well-known couplingpromoters, such N-hydroxysuccinimide, 1-hydroxybenzotriazole (HOBT),1-hydroxy-7-azabenzotriazole (HOAT), N,N-dimethylaminopyridine (DMAP)and the like, can be employed in this reaction. Typically, this couplingreaction is conducted at a temperature ranging from about 0° C. to about60° C. for about 1 to about 72 hours in an inert diluent, such as THF orDMF. In certain instances, Compound S-202 reacts with Compound S-301 toform Compound S-302 in the presence of HATU.

With continued reference to Scheme 3, Compound S-302 is transformed intoCompound S-303 with removal of the amino protecting group. Conditions toremove amino groups can be found in Greene and Wuts. When PG² is a Bocgroup, the protecting group can be removed with acidic conditions, suchas treatment with hydrochloric acid or trifluoroacetic acid.

A representative synthesis for Compound S-401 is shown in Scheme 4. InScheme 4, for Compound KC-8, R^(a) is hydroxyl; n is 3; the first andthird geminal R¹ and R² are hydrogen; the second geminal R¹ and R² aremethyl; R⁵ is methyl; R⁶ is the side chain of arginine; R⁷ is the sidechain of glycine; and R⁸ is malonyl group.

In Scheme 4, Compound S-400 is a commercially available startingmaterial. Alternatively, Compound S-400 can be synthesized via a varietyof different synthetic routes using commercially available startingmaterials and/or starting materials prepared by conventional syntheticmethods.

With reference to Scheme 4, Compound S-303 reacts with Compound S-400 toform Compound S-401 in a peptide coupling reaction. A peptide couplingreaction typically employs a conventional peptide coupling reagent andis conducted under conventional coupling reaction conditions, typicallyin the presence of a trialkylamine, such as ethyldiisopropylamine ordiisopropylethylamine (DIEA). Suitable coupling reagents for useinclude, by way of example, carbodiimides, such asethyl-3-(3-dimethylamino)propylcarbodiimide (EDC),dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) and thelike, and other well-known coupling reagents, such asN,N′-carbonyldiimidazole, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline(EEDQ), benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP),0-(7-azabenzotriazol-1-yl)-N,N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) and the like. Optionally, well-known couplingpromoters, such N-hydroxysuccinimide, 1-hydroxybenzotriazole (HOBT),1-hydroxy-7-azabenzotriazole (HOAT), N,N-dimethylaminopyridine (DMAP)and the like, can be employed in this reaction. Typically, this couplingreaction is conducted at a temperature ranging from about 0° C. to about60° C. for about 1 to about 72 hours in an inert diluent, such as THF orDMF. In certain instances, Compound S-303 reacts with Compound S-400 toform Compound S-401 in the presence of HATU.

In certain instances in Scheme 4, when Compound S-400 is reacted withCompound S-303 with R⁸ as hydrogen, the R⁸ group as a malonyl group isadded after the amino acid coupling reaction. A malonyl group can beattached via a reaction with mono-tert-butyl malonate. Reaction usingmono-tert-butyl malonate can be aided with use of activation reagents,such as symmetric anhydrides,O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate(HBTU), dicyclohexylcarbodiimide (DCC) diisopropylcarbodiimide(DIC)/1-hydroxybenzotriazole (HOBt), andbenzotriazole-1-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate(BOP). A malonyl group can also be attached using N-carboxymethylmalonicacid tert-butyl ester.

With continued reference to Scheme 4, removal of other protecting groupscan be performed if other protecting groups were used, such asprotecting groups present on the R⁶ moiety. Conditions for removal ofother protecting groups depend on the identity of the protecting groupand are known to those skilled in the art. The conditions can also befound in Greene and Wuts.

Further Representative Synthetic Schemes

A representative synthesis for Compound S-503 is shown in Scheme 5. InScheme 5, for Compound KC-8, n is 3; the first and third geminal R¹ andR² are hydrogen; the second geminal R¹ and R² are methyl; R⁵ is methyl;and PG¹ and PG² are optional amino protecting groups.

In Scheme 5, Compound S-500 is a commercially available startingmaterial. Alternatively, Compound S-500 can be synthesized via a varietyof different synthetic routes using commercially available startingmaterials and/or starting materials prepared by conventional syntheticmethods.

With continued reference to Scheme 5, Compound S-500 is protected at theamino group to form Compound S-501, wherein PG¹ and PG² are aminoprotecting groups. Amino protecting groups can be found in T. W. Greeneand P. G. M. Wuts, “Protective Groups in Organic Synthesis”, Fourthedition, Wiley, New York 2006. Representative amino-protecting groupsinclude, but are not limited to, formyl groups; acyl groups, for examplealkanoyl groups, such as acetyl; alkoxycarbonyl groups, such astert-butoxycarbonyl (Boc); arylmethoxycarbonyl groups, such asbenzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc);arylmethyl groups, such as benzyl (Bn), trityl (Tr), and1,1-di-(4′-methoxyphenyl)methyl; silyl groups, such as trimethylsilyl(TMS) and tert-butyldimethylsilyl (TBS); and the like.

In certain embodiments, PG¹ and PG² are Boc groups. Conditions forforming Boc groups on Compound S-501 can be found in Greene and Wuts.One method is reaction of Compound S-500 with di-tert-butyl dicarbonate.The reaction can optionally be run in the presence of an activatingagent, such as DMAP.

With continued reference to Scheme 5, the carboxybenzyl group onCompound S-501 is deprotected to form Compound S-502. Conditions toremove the carboxybenzyl group can be found in Greene and Wuts. Methodsto remove the carboxybenzyl group include hydrogenolysis of CompoundS-501 or treatment of Compound S-501 with HBr. One method to remove thecarboxybenzyl group is reaction of Compound S-501 with hydrogen andpalladium.

With continued reference to Scheme 5, Compound S-502 is reacted withphosgene to form Compound S-503. Reaction with phosgene forms an acylchloride on the amino group of Compound S-502. Other reagents can act assubstitutes for phosgene, such as diphosgene or triphosgene.

A representative synthesis for Compound S-602 is shown in Scheme 6. InScheme 6, for Compound KC-8, R^(a) is hydroxyl; n is 3; the first andthird geminal R¹ and R² are hydrogen; the second geminal R¹ and R² aremethyl; R⁵ is methyl; and PG¹ and PG² are optional amino protectinggroups.

In Scheme 6, Compound S-600 is a commercially available startingmaterial. Alternatively, Compound S-600 can be semi-syntheticallyderived from natural materials or synthesized via a variety of differentsynthetic routes using commercially available starting materials and/orstarting materials prepared by conventional synthetic methods.

With continued reference to Scheme 6, Compound S-600 is reacted withCompound S-503 to form Compound S-601. In this reaction, the enolate ofCompound S-600 reacts with the acyl chloride of Compound S-503 to form acarbamate.

With continued reference to Scheme 6, the optional protecting groups PG¹and PG² are removed from Compound S-601 to form Compound S-602.Conditions to remove amino groups can be found in Greene and Wuts. WhenPG¹ and/or PG² are Boc groups, the protecting groups can be removed withacidic conditions, such as treatment with hydrochloric acid ortrifluoroacetic acid.

Compound S-602 can be used as in the above schemes, such as Schemes 3and 4, to prepare Compound KC-8.

As described in more detail herein, the disclosure provides processesand intermediates useful for preparing compounds of the presentdisclosure or a salt or solvate or stereoisomer thereof. Accordingly,the present disclosure provides a process of preparing a compound of thepresent disclosure, the process involves:

contacting a compound of formula:

with a compound of formula

wherein R^(a) is hydroxyl; n is 3; the first and third geminal R¹ and R²are hydrogen; the second geminal R¹ and R² are methyl; R⁵ is methyl; andPG¹ is an amino protecting group, in the presence of a carbamate-formingreagent.

Accordingly and as described in more detail herein, the presentdisclosure provides a process of preparing a compound of the presentdisclosure, the process involves:

contacting a compound of formula:

with a compound of formula

wherein R^(a) is hydroxyl; n is 3; the first and third geminal R¹ and R²are hydrogen; the second geminal R¹ and R² are methyl; R⁵ is methyl; R⁶is the side chain of arginine; and PG² is an amino protecting group.

Accordingly and as described in more detail herein, the presentdisclosure provides a process of preparing a compound of the presentdisclosure, the process involves: contacting a compound of formula:

with a compound of formula

wherein R^(a) is hydroxyl; n is 3; the first and third geminal R¹ and R²are hydrogen; the second geminal R¹ and R² are methyl; R⁵ is methyl; R⁶is the side chain of arginine; R⁷ is the side chain of glycine; and R⁸is malonyl.

In one instance, the above process can further involve a step of forminga salt of a compound of the present disclosure. Embodiments are directedto the other processes described herein; and to the product prepared byany of the processes described herein.

Trypsin Inhibitors

As disclosed herein, the present disclosure also provides pharmaceuticalcompositions, and their methods of use, where the pharmaceuticalcompositions comprise a prodrug, Compound KC-8, that provides controlledrelease of oxycodone via enzyme cleavage followed by intramolecularcyclization, and a trypsin inhibitor that interacts with the enzyme thatmediates the enzymatically-mediated release of oxycodone from theprodrug so as to attenuate enzymatic cleavage of the prodrug. Suchdisclosure provides for the enzyme being trypsin.

As used herein, the term “trypsin inhibitor” refers to any agent capableof inhibiting the action of trypsin on a substrate. The term “trypsininhibitor” also encompasses salts of trypsin inhibitors. The ability ofan agent to inhibit trypsin can be measured using assays well known inthe art. For example, in a typical assay, one unit corresponds to theamount of inhibitor that reduces the trypsin activity by onebenzoyl-L-arginine ethyl ester unit (BAEE-U). One BAEE-U is the amountof enzyme that increases the absorbance at 253 nm by 0.001 per minute atpH 7.6 and 25° C. See, for example, K. Ozawa, M. Laskowski, 1966, J.Biol. Chem. 241, 3955 and Y. Birk, 1976, Meth. Enzymol. 45, 700. Incertain instances, a trypsin inhibitor can interact with an active siteof trypsin, such as the S1 pocket and the S3/4 pocket. The S1 pocket hasan aspartate residue which has affinity for a positively charged moiety.The S3/4 pocket is a hydrophobic pocket. The disclosure provides forspecific trypsin inhibitors and non-specific serine protease inhibitors.

There are many trypsin inhibitors known in the art, both those specificto trypsin and those that inhibit trypsin and other proteases such aschymotrypsin. The disclosure provides for trypsin inhibitors that areproteins, peptides, and small molecules. The disclosure provides fortrypsin inhibitors that are irreversible inhibitors or reversibleinhibitors. The disclosure provides for trypsin inhibitors that arecompetitive inhibitors, non-competitive inhibitors, or uncompetitiveinhibitors. The disclosure provides for natural, synthetic orsemi-synthetic trypsin inhibitors.

Trypsin inhibitors can be derived from a variety of animal or vegetablesources: for example, soybean, corn, lima and other beans, squash,sunflower, bovine and other animal pancreas and lung, chicken and turkeyegg white, soy-based infant formula, and mammalian blood. Trypsininhibitors can also be of microbial origin: for example, antipain; see,for example, H. Umezawa, 1976, Meth. Enzymol. 45, 678. A trypsininhibitor can also be an arginine or lysine mimic or other syntheticcompound: for example arylguanidine, benzamidine,3,4-dichloroisocoumarin, diisopropylfluorophosphate, gabexate mesylate,phenylmethanesulfonyl fluoride, or substituted versions or analogsthereof. In certain embodiments, trypsin inhibitors comprise acovalently modifiable group, such as a chloroketone moiety, an aldehydemoiety, or an epoxide moiety. Other examples of trypsin inhibitors areaprotinin, camostat and pentamidine.

As used herein, an arginine or lysine mimic is a compound that iscapable of binding to the P¹ pocket of trypsin and/or interfering withtrypsin active site function. The arginine or lysine mimic can be acleavable or non-cleavable moiety.

In one embodiment, the trypsin inhibitor is derived from soybean.Trypsin inhibitors derived from soybean (Glycine max) are readilyavailable and are considered to be safe for human consumption. Theyinclude, but are not limited to, SBTI, which inhibits trypsin, andBowman-Birk inhibitor, which inhibits trypsin and chymotrypsin. Suchtrypsin inhibitors are available, for example from Sigma-Aldrich, St.Louis, Mo., USA.

It will be appreciated that the pharmaceutical composition according tothe embodiments may further comprise one or more other trypsininhibitors.

As stated above, a trypsin inhibitor can be an arginine or lysine mimicor other synthetic compound. In certain embodiments, the trypsininhibitor is an arginine mimic or a lysine mimic, wherein the argininemimic or lysine mimic is a synthetic compound.

Certain trypsin inhibitors include compounds of formula:

wherein:

Q¹ is selected from —O-Q⁴ or -Q⁴-COOH, where Q⁴ is C₁-C₄ alkyl;

Q² is N or CH; and

Q³ is aryl or substituted aryl.

Certain trypsin inhibitors include compounds of formula:

wherein:

Q⁵ is —C(O)—COOH or —NH-Q⁶-Q⁷-SO₂—C₆H₅, where

Q⁶ is —(CH₂)_(p)—COOH;

Q⁷ is —(CH₂)—C₆H₅;

Q⁸ is NH;

n is a number from zero to two;

o is zero or one;

p is an integer from one to three; and

r is an integer from one to three.

Certain trypsin inhibitors include compounds of formula:

wherein:

Q⁵ is —C(O)—COOH or —NH-Q⁶-Q⁷-SO₂—C₆H₅, where

Q⁶ is —(CH₂)_(p)—COOH;

Q⁷ is —(CH₂), C₆H₅; and

p is an integer from one to three; and

r is an integer from one to three.

Certain trypsin inhibitors include the following:

Compound 101

(S)-ethyl 4-(5-guanidino-2- (naphthalene-2- sulfonamido)pentanoyl)piper-azine-1-carboxylate Compound 102

(S)-ethyl 4-(5-guanidino-2- (2,4,6- triisopropylphenylsulfon-amido)pentanoyl)piperazine- 1-carboxylate Compound 103

(S)-ethyl 1-(5-guanidino-2- (naphthalene-2- sulfonamido)pentanoyl)piper-idine-4-carboxylate Compound 104

(S)-ethyl 1-(5-guanidino-2- (2,4,6- triisopropylphenylsulfon-amido)pentanoyl)piperidine- 4-carboxylate Compound 105

(S)-6-(4-(5-guanidino-2- (naphthalene-2- sulfonamido)pentanoyl)piper-azin-1-yl)-6-oxohexanoic acid Compound 106

4-aminobenzimidamide (also 4-aminobenzamidine) Compound 107

3-(4- carbamimidoylphenyl)-2- oxopropanoic acid Compound 108

(S)-5-(4- carbamimidoylbenzylamino)- 5-oxo-4-((R)-4-phenyl- 2-(phenylmethylsulfonamido)- butanamido)pentanoic acid Compound 109

6- carbamimidoylnaphthalen- 2-yl 4- (diaminomethyleneamino) benzoateCompound 110

4,4′-(pentane-1,5- diylbis(oxy))dibenzimid- amide

A description of methods to prepare Compound 101, Compound 102, Compound103, Compound 104, Compound 105, Compound 107, and Compound 108 isprovided in PCT International Publication Number WO 2010/045599A1,published 22 Apr. 2010, which is incorporated herein by reference in itsentirety. Compound 106, Compound 109, and Compound 110 are commerciallyavailable, e.g., from Sigma-Aldrich, St. Louis, Mo., USA.

In certain embodiments, the trypsin inhibitor is SBTI, BBSI, Compound101, Compound 106, Compound 108, Compound 109, or Compound 110. Incertain embodiments, the trypsin inhibitor is camostat.

In certain embodiments, the trypsin inhibitor is a compound of formulaT-I:

wherein

A represents a group of the following formula:

R^(t9) and R^(t10) each represents independently a hydrogen atom or aC₁₋₄ alkyl group,

R^(t8) represents a group selected from the following formulae:

wherein R each represents independently

(1) a hydrogen atom,

(2) a phenyl group,

(3) a C₁₋₄ alkyl group substituted by a phenyl group,

(4) a C₁₋₁₀ alkyl group,

(5) a C₁₋₁₀ alkoxyl group,

(6) a C₂₋₁₀ alkenyl group having 1 to 3 double bonds,

(7) a C₂₋₁₀ alkynyl group having 1 to 2 triple bonds,

(8) a group of formula: R^(t15)—C(O)XR^(t16),

wherein R^(t15) represents a single bond or a C₁₋₈ alkylene group,

X represents an oxygen atom or an NH-group, and

R^(t16) represents a hydrogen atom, a C₁₋₄ alkyl group, a phenyl groupor a C₁₋₄ alkyl group substituted by a phenyl group, or

(9) a C₃₋₇ cycloalkyl group;

the structure

represents a 4-7 membered monocyclic hetero-ring containing 1 to 2nitrogen or oxygen atoms,

R^(t14) represents a hydrogen atom, a C₁₋₄ alkyl group substituted by aphenyl group or a group of formula: COOR^(t17), wherein R^(t17)represents a hydrogen atom, a C₁₋₄ alkyl group or a C₁₋₄ alkyl groupsubstituted by a phenyl group;

provided that R^(t11), R^(t12) and R^(t13) do not representsimultaneously hydrogen atoms;

or nontoxic salts, acid addition salts or hydrates thereof.

In certain embodiments, the trypsin inhibitor is a compound selectedfrom the following:

In certain embodiments, the trypsin inhibitor is a compound of formulaT-II:

wherein

X is NH;

n is zero or one; and

R^(t1) is selected from hydrogen, halogen, nitro, alkyl, substitutedalkyl, alkoxy, carboxyl, alkoxycarbonyl, acyl, aminoacyl, guanidine,amidino, carbamide, amino, substituted amino, hydroxyl, cyano and—(CH₂)_(m)—C(O)—O—(CH₂)_(m)—C(O)—N—R^(n1)R^(n2), wherein each m isindependently zero to 2; and R^(n1) and R^(n2) are independentlyselected from hydrogen and C₁₋₄ alkyl.

In certain embodiments, in formula T-II, R^(t1) is guanidino or amidino.

In certain embodiments, in formula T-II, R^(t1) is—(CH₂)_(m)—C(O)—O—(CH₂)_(m)—C(O)—N—R^(n1)R^(n2), wherein m is one andR^(n1) and R^(n2) are methyl.

In certain embodiments, the trypsin inhibitor is a compound of formulaT-III:

wherein

X is NH;

n is zero or one;

L^(t1) is selected from —C(O)—O—; —O—C(O)—; —O—(CH₂)_(m)—O—;—OCH₂—Ar^(t2)—CH₂O—; —C(O)—NR^(t3)—; and —NR^(t3)—C(O)—;

R^(t3) is selected from hydrogen, C₁₋₆ alkyl, and substituted C₁₋₆alkyl;

Ar^(t1) and Ar^(t2) are independently a substituted or unsubstitutedaryl group;

m is a number from 1 to 3; and

R^(t2) is selected from hydrogen, halogen, nitro, alkyl, substitutedalkyl, alkoxy, carboxyl, alkoxycarbonyl, acyl, aminoacyl, guanidine,amidino, carbamide, amino, substituted amino, hydroxyl, cyano and—(CH₂)_(m)—C(O)—O—(CH₂)_(m)—C(O)_N_R^(n1)R^(n2), wherein each m isindependently zero to 2; and R^(n1) and R^(n2) are independentlyselected from hydrogen and C₁₋₄ alkyl.

In certain embodiments, in formula T-III, R^(t2) is guanidino oramidino.

In certain embodiments, in formula T-III, R^(t2) is—(CH₂)_(m)—C(O)—O—(CH₂)_(m)—C(O)—N—R^(n1)R^(n2), wherein m is one andR^(n1) and R^(n2) are methyl.

In certain embodiments, the trypsin inhibitor is a compound of formulaT-IV:

wherein

each X is NH;

each n is independently zero or one;

L^(t1) is selected from —C(O)—O—; —O—C(O)—; —O—(CH₂)_(m)—O—;—OCH₂—Ar^(t2)—CH₂O—; —C(O)—NR^(t3)—; and —NR^(t3)—C(O)—;

R^(t3) is selected from hydrogen, C₁₋₆ alkyl, and substituted C₁₋₆alkyl;

Ar^(t1) and Ar^(t2) are independently a substituted or unsubstitutedaryl group; and

m is a number from 1 to 3.

In certain embodiments, in formula T-IV, Ar^(t1) or Ar^(t2) is phenyl.

In certain embodiments, in formula T-IV, Ar^(t1) or Ar^(t2) is naphthyl.

In certain embodiments, the trypsin inhibitor is Compound 109.

In certain embodiments, the trypsin inhibitor is

In certain embodiments, the trypsin inhibitor is Compound 110 or abis-arylamidine variant thereof; see, for example, J. D. Geratz, M.C.-F. Cheng and R. R. Tidwell (1976) J. Med. Chem. 19, 634-639.

It is to be appreciated that the invention also includes inhibitors ofother enzymes involved in protein assimilation that can be used incombination with Compound KC-8 to attenuate release of oxycodone fromthe prodrug.

Combinations of Prodrug and Trypsin Inhibitor

As discussed above, the present disclosure provides pharmaceuticalcompositions which comprise a trypsin inhibitor and Compound KC-8, aketone-modified oxycodone prodrug, that comprises a promoiety comprisinga trypsin-cleavable moiety that, when cleaved, facilitates release ofoxycodone. Examples of compositions containing Compound KC-8 and atrypsin inhibitor are described below.

The embodiments provide a pharmaceutical composition, which comprises acompound of Formulae T-I to T-IV and Compound KC-8, or apharmaceutically acceptable salt thereof. The embodiments provide apharmaceutical composition, which comprises Compound 109 and CompoundKC-8, or a pharmaceutically acceptable salt thereof.

Certain embodiments provide for a combination of Compound KC-8 and atrypsin inhibitor, in which the trypsin inhibitor is shown in thefollowing table.

Prodrug Trypsin inhibitor Compound KC-8 SBTI Compound KC-8 BBSI CompoundKC-8 Compound 101 Compound KC-8 Compound 106 Compound KC-8 Compound 108Compound KC-8 Compound 109 Compound KC-8 Compound 110 Compound KC-8camostatCombinations of Compound KC-8 and Other Drugs

The disclosure provides for Compound KC-8 and a further prodrug or drugincluded in a pharmaceutical composition. Such a prodrug or drug wouldprovide additional analgesia or other benefits. Examples includeopioids, acetaminophen, non-steroidal anti-inflammatory drugs (NSAIDs)and other analgesics. In one embodiment, Compound KC-8 would be combinedwith an opioid antagonist prodrug or drug. Other examples include drugsor prodrugs that have benefits other than, or in addition to, analgesia.The embodiments provide a pharmaceutical composition, which comprisesCompound KC-8 and acetaminophen, or a pharmaceutically acceptable saltthereof.

Such compositions can also comprise a trypsin inhibitor. In certainembodiments, the trypsin inhibitor is selected from SBTI, BBSI, Compound101, Compound 106, Compound 108, Compound 109, and Compound 110. Incertain embodiments, the trypsin inhibitor is Compound 109. In certainembodiments, the trypsin inhibitor is camostat.

In certain embodiments, a pharmaceutical composition can compriseCompound KC-8, a non-opioid drug and at least one opioid or opioidprodrug.

Pharmaceutical Compositions and Methods of Use

As disclosed herein, the embodiments provide a composition, whichcomprisesN-1-[3-(oxycodone-6-enol-carbonyl-methyl-amino)-2,2-dimethyl-propylamine]-arginine-glycine-malonicacid, Compound KC-8. The pharmaceutical composition according to theembodiments can further comprise a pharmaceutically acceptable carrier.The composition is conveniently formulated in a form suitable for oral(including buccal and sublingual) administration, for example as atablet, capsule, thin film, powder, suspension, solution, syrup,dispersion or emulsion. The composition can contain componentsconventional in pharmaceutical preparations, e.g. one or more carriers,binders, lubricants, excipients (e.g., to impart controlled releasecharacteristics), pH modifiers, sweeteners, bulking agents, coloringagents or further active agents.

Patients can be humans, and also other mammals, such as livestock, zooanimals and companion animals, such as a cat, dog or horse.

In another aspect, the embodiments provide a pharmaceutical compositionas described hereinabove for use in the treatment of pain. Thepharmaceutical composition according to the embodiments is useful, forexample, in the treatment of a patient suffering from, or at risk ofsuffering from, pain. Accordingly, the present disclosure providesmethods of treating or preventing pain in a subject, the methodsinvolving administering to the subject a disclosed composition. Thepresent disclosure provides for a disclosed composition for use intherapy or prevention or as a medicament. The present disclosure alsoprovides the use of a disclosed composition for the manufacture of amedicament, especially for the manufacture of a medicament for thetreatment or prevention of pain.

The compositions of the present disclosure can be used in the treatmentor prevention of pain including, but not limited to include, acute pain,chronic pain, neuropathic pain, acute traumatic pain, arthritic pain,osteoarthritic pain, rheumatoid arthritic pain, muscular skeletal pain,post-dental surgical pain, dental pain, myofascial pain, cancer pain,visceral pain, diabetic pain, muscular pain, post-herpetic neuralgicpain, chronic pelvic pain, endometriosis pain, pelvic inflammatory painand child birth related pain. Acute pain includes, but is not limitedto, acute traumatic pain or post-surgical pain. Chronic pain includes,but is not limited to, neuropathic pain, arthritic pain, osteoarthriticpain, rheumatoid arthritic pain, muscular skeletal pain, dental pain,myofascial pain, cancer pain, diabetic pain, visceral pain, muscularpain, post-herpetic neuralgic pain, chronic pelvic pain, endometriosispain, pelvic inflammatory pain and back pain.

The present disclosure also provides use of Compound KC-8 in thetreatment of pain. The present disclosure also provides use of CompoundKC-8 in the prevention of pain.

The present disclosure provides use of Compound KC-8 in the manufactureof a medicament for treatment of pain. The present disclosure providesuse of Compound KC-8 in the manufacture of a medicament for preventionof pain.

In another aspect, the embodiments provide a method of treating pain ina patient in need thereof, which comprises administering an effectiveamount of a pharmaceutical composition as described hereinabove. Inanother aspect, the embodiments provide a method of preventing pain in apatient in need thereof, which comprises administering an effectiveamount of a pharmaceutical composition as described hereinabove.

The amount of composition disclosed herein to be administered to apatient to be effective (i.e. to provide blood levels of oxycodonesufficient to be effective in the treatment or prophylaxis of pain) willdepend upon the bioavailability of the particular composition, thesusceptibility of the particular composition to enzyme activation in thegut, as well as other factors, such as the species, age, weight, sex,and condition of the patient, manner of administration and judgment ofthe prescribing physician. If the composition also comprises a trypsininhibitor, the amount of composition disclosed herein to be administeredto a patient would also depend on the amount and potency of trypsininhibitor present in the composition. In general, the composition dosecan be such that Compound KC-8 is in the range of from 0.01 milligramsprodrug per kilogram to 20 milligrams prodrug per kilogram (mg/kg) bodyweight. For example, a composition comprising Compound KC-8 can beadministered at a dose equivalent to administering free oxycodone in therange of from 0.02 to 0.5 mg/kg body weight or 0.01 mg/kg to 10 mg/kgbody weight or 0.01 to 2 mg/kg body weight. In one embodiment, thecomposition can be administered at a dose such that the level ofoxycodone achieved in the blood is in the range of from 0.5 ng/ml to 10ng/ml.

As disclosed above, the present disclosure also provides pharmaceuticalcompositions which comprise a trypsin inhibitor and Compound KC-8, aphenol-modified oxycodone prodrug, that comprises a promoiety comprisinga trypsin-cleavable moiety that, when cleaved, facilitates release ofoxycodone. In such pharmaceutical compositions, the amount of a trypsininhibitor to be administered to the patient to be effective (i.e. toattenuate release of oxycodone when administration of Compound KC-8alone would lead to overexposure of oxycodone) will depend upon theeffective dose of Compound KC-8 and the potency of the particulartrypsin inhibitor, as well as other factors, such as the species, age,weight, sex and condition of the patient, manner of administration andjudgment of the prescribing physician. In general, the dose of trypsininhibitor can be in the range of from 0.05 mg to 50 mg per mg ofCompound KC-8. In a certain embodiment, the dose of trypsin inhibitorcan be in the range of from 0.001 mg to 50 mg per mg of Compound KC-8.In one embodiment, the dose of trypsin inhibitor can be in the range offrom 0.01 nanomoles to 100 micromoles per micromole of Compound KC-8.

Representative Embodiments of Dose Units of Prodrug Compound KC-8 andTrypsin Inhibitor Having a Desired Pharmacokinetic Profile

The embodiments include a composition that comprises (a) a prodrugcomprising oxycodone covalently bound through the enolic oxygen to apromoiety comprising a trypsin-cleavable moiety, wherein cleavage of thetrypsin-cleavable moiety by trypsin mediates release of oxycodone,wherein the prodrug is Compound KC-8 and (b) a trypsin inhibitor thatinteracts with the trypsin that mediates enzymatically-controlledrelease of oxycodone from the prodrug following ingestion of thecomposition.

The embodiments include a dose unit comprising a composition, such as apharmaceutical composition, comprising Compound KC-8, a ketone-modifiedprodrug, and a trypsin inhibitor, where Compound KC-8 and trypsininhibitor are present in the dose unit in an amount effective to providefor a pre-selected pharmacokinetic (PK) profile following ingestion. Infurther embodiments, the pre-selected PK profile comprises at least onePK parameter value that is less than the PK parameter value of oxycodonereleased following ingestion of an equivalent dosage of Compound KC-8 inthe absence of inhibitor. In further embodiments, the PK parameter valueis selected from an oxycodone Cmax value, an oxycodone exposure value,and a (1/oxycodone Tmax) value.

In certain embodiments, the dose unit provides for a pre-selected PKprofile following ingestion of at least two dose units. In relatedembodiments, the pre-selected PK profile of such dose units is modifiedrelative to the PK profile following ingestion of an equivalent dosageof Compound KC-8 without inhibitor. In related embodiments, such a doseunit provides that ingestion of an increasing number of the dose unitsprovides for a linear PK profile. In related embodiments, such a doseunit provides that ingestion of an increasing number of the dose unitsprovides for a nonlinear PK profile. In related embodiments, the PKparameter value of the PK profile of such a dose unit is selected froman oxycodone Cmax value, a (1/oxycodone Tmax) value, and an oxycodoneexposure value.

The embodiments include methods for treating a patient comprisingadministering any of the compositions, such as pharmaceuticalcompositions, comprising Compound KC-8 and a trypsin inhibitor or doseunits described herein to a patient in need thereof. The embodimentsinclude methods to reduce side effects of a therapy comprisingadministering any of such compositions, e.g., pharmaceuticalcompositions, or dose units described herein to a patient in needthereof. The embodiments include methods of improving patient compliancewith a therapy prescribed by a clinician comprising directingadministration of any of such compositions, e.g., pharmaceuticalcompositions, or dose units described herein to a patient in needthereof. Such embodiments can provide for improved patient compliancewith a prescribed therapy as compared to patient compliance with aprescribed therapy using drug and/or using prodrug without inhibitor ascompared to prodrug with inhibitor.

The embodiments include methods of reducing risk of unintended overdoseof oxycodone comprising directing administration of any of suchcompositions, e.g., pharmaceutical compositions, or dose units describedherein to a patient in need of treatment.

The embodiments include methods of making a dose unit comprisingcombining Compound KC-8 and a trypsin inhibitor in a dose unit, whereinCompound KC-8 and trypsin inhibitor are present in the dose unit in anamount effective to attenuate release of oxycodone from Compound KC-8.

The embodiments include methods of deterring misuse or abuse of multipledose units of Compound KC-8 comprising combining Compound KC-8 and atrypsin inhibitor in a dose unit, wherein Compound KC-8 and trypsininhibitor are present in the dose unit in an amount effective toattenuate release of oxycodone from Compound KC-8 such that ingestion ofmultiples of dose units by a patient does not provide a proportionalrelease of oxycodone. In further embodiments, release of drug isdecreased compared to release of drug by an equivalent dosage of prodrugin the absence of inhibitor.

One embodiment is a method for identifying a trypsin inhibitor andprodrug Compound KC-8 suitable for formulation in a dose unit. Such amethod can be conducted as, for example, an in vitro assay, an in vivoassay, or an ex vivo assay.

The embodiments include methods for identifying a trypsin inhibitor andprodrug Compound KC-8 suitable for formulation in a dose unit comprisingcombining prodrug Compound KC-8, a trypsin inhibitor, and trypsin in areaction mixture, and detecting prodrug conversion, wherein a decreasein prodrug conversion in the presence of the trypsin inhibitor ascompared to prodrug conversion in the absence of the trypsin inhibitorindicates the trypsin inhibitor and prodrug Compound KC-8 are suitablefor formulation in a dose unit.

The embodiments include methods for identifying a trypsin inhibitor andprodrug Compound KC-8 suitable for formulation in a dose unit comprisingadministering to an animal a trypsin inhibitor and prodrug Compound KC-8and detecting prodrug conversion, wherein a decrease in oxycodoneconversion in the presence of the trypsin inhibitor as compared tooxycodone conversion in the absence of the trypsin inhibitor indicatesthe trypsin inhibitor and prodrug Compound KC-8 are suitable forformulation in a dose unit. In certain embodiments, administeringcomprises administering to the animal increasing doses of inhibitorco-dosed with a selected fixed dose of prodrug. Detecting prodrugconversion can facilitate identification of a dose of inhibitor and adose of prodrug that provides for a pre-selected pharmacokinetic (PK)profile. Such methods can be conducted as, for example, an in vivo assayor an ex vivo assay.

The embodiments include methods for identifying a trypsin inhibitor andprodrug Compound KC-8 suitable for formulation in a dose unit comprisingadministering to an animal tissue a trypsin inhibitor and prodrugCompound KC-8 and detecting prodrug conversion, wherein a decrease inprodrug conversion in the presence of the trypsin inhibitor as comparedto prodrug conversion in the absence of the trypsin inhibitor indicatesthe trypsin inhibitor and prodrug Compound KC-8 are suitable forformulation in a dose unit.

Dose Units of Prodrug Compound KC-8 and Trypsin Inhibitor Having aDesired Pharmacokinetic Profile

The present disclosure provides dose units of prodrug and inhibitor thatcan provide for a desired pharmacokinetic (PK) profile. Dose units canprovide a modified PK profile compared to a reference PK profile asdisclosed herein. It will be appreciated that a modified PK profile canprovide for a modified pharmacodynamic (PD) profile. Ingestion ofmultiples of such a dose unit can also provide a desired PK profile.

Unless specifically stated otherwise, “dose unit” as used herein refersto a combination of a trypsin-cleavable prodrug and a trypsin inhibitor.A “single dose unit” is a single unit of a combination of atrypsin-cleavable prodrug and a trypsin inhibitor, where the single doseunit provide a therapeutically effective amount of drug (i.e., asufficient amount of drug to effect a therapeutic effect, e.g., a dosewithin the respective drug's therapeutic window, or therapeutic range).“Multiple dose units” or “multiples of a dose unit” or a “multiple of adose unit” refers to at least two single dose units.

As used herein, a “PK profile” refers to a profile of drug concentrationin blood or plasma. Such a profile can be a relationship of drugconcentration over time (i.e., a “concentration-time PK profile”) or arelationship of drug concentration versus number of doses ingested(i.e., a “concentration-dose PK profile”.) A PK profile is characterizedby PK parameters.

As used herein, a “PK parameter” refers to a measure of drugconcentration in blood or plasma, such as: 1) “drug Cmax”, the maximumconcentration of drug achieved in blood or plasma; 2) “drug T_(max)”,the time elapsed following ingestion to achieve Cmax; and 3) “drugexposure”, the total concentration of drug present in blood or plasmaover a selected period of time, which can be measured using the areaunder the curve (AUC) of a time course of drug release over a selectedperiod of time (t). Modification of one or more PK parameters providesfor a modified PK profile.

For purposes of describing the features of dose units of the presentdisclosure, “PK parameter values” that define a PK profile include drugCmax (e.g., oxycodone Cmax), total drug exposure (e.g., area under thecurve) (e.g., oxycodone exposure) and 1/(drug Tmax) (such that adecreased 1/Tmax is indicative of a delay in Tmax relative to areference Tmax) (e.g., 1/oxycodone Tmax). Thus a decrease in a PKparameter value relative to a reference PK parameter value can indicate,for example, a decrease in drug Cmax, a decrease in drug exposure,and/or a delayed T_(max).

Dose units of the present disclosure can be adapted to provide for amodified PK profile, e.g., a PK profile that is different from thatachieved from dosing a given dose of prodrug in the absence of inhibitor(i.e., without inhibitor). For example, dose units can provide for atleast one of decreased drug Cmax, delayed drug Tmax and/or decreaseddrug exposure compared to ingestion of a dose of prodrug in the sameamount but in the absence of inhibitor. Such a modification is due tothe inclusion of an inhibitor in the dose unit.

As used herein, “a pharmacodynamic (PD) profile” refers to a profile ofthe efficacy of a drug in a patient (or subject or user), which ischaracterized by PD parameters. “PD parameters” include “drug Emax” (themaximum drug efficacy), “drug EC50” (the concentration of drug at 50% ofthe Emax), and side effects.

FIG. 1 is a schematic illustrating an example of the effect ofincreasing inhibitor concentrations upon the PK parameter drug Cmax fora fixed dose of prodrug. At low concentrations of inhibitor, there maybe no detectable effect on drug release, as illustrated by the plateauportion of the plot of drug Cmax (Y axis) versus inhibitor concentration(X axis). As inhibitor concentration increases, a concentration isreached at which drug release from prodrug is attenuated, causing adecrease in, or suppression of, drug Cmax. Thus, the effect of inhibitorupon a prodrug PK parameter for a dose unit of the present disclosurecan range from undetectable, to moderate, to complete inhibition (i.e.,no detectable drug release).

A dose unit can be adapted to provide for a desired PK profile (e.g., aconcentration-time PK profile) following ingestion of a single dose. Adose unit can be adapted to provide for a desired PK profile (e.g., aconcentration-dose PK profile) following ingestion of multiple doseunits (e.g., at least 2, at least 3, at least 4 or more dose units).

Dose Units Providing Modified PK Profiles

A combination of a prodrug and an inhibitor in a dose unit can provide adesired (or “pre-selected”) PK profile (e.g., a concentration-time PKprofile) following ingestion of a single dose. The PK profile of such adose unit can be characterized by one or more of a pre-selected drugCmax, a pre-selected drug Tmax or a pre-selected drug exposure. The PKprofile of the dose unit can be modified compared to a PK profileachieved from the equivalent dosage of prodrug in the absence ofinhibitor (i.e., a dose that is the same as the dose unit except that itlacks inhibitor).

A modified PK profile can have a decreased PK parameter value relativeto a reference PK parameter value (e.g., a PK parameter value of a PKprofile following ingestion of a dosage of prodrug that is equivalent toa dose unit except without inhibitor). For example, a dose unit canprovide for a decreased drug Cmax, decreased drug exposure, and/ordelayed drug Tmax.

FIG. 2 presents schematic graphs showing examples of modifiedconcentration-time PK profiles of a single dose unit. Panel A is aschematic of drug concentration in blood or plasma (Y axis) following aperiod of time (X axis) after ingestion of prodrug in the absence orpresence of inhibitor. The solid, top line in Panel A provides anexample of drug concentration following ingestion of prodrug withoutinhibitor. The dashed, lower line in Panel A represents drugconcentration following ingestion of the same dose of prodrug withinhibitor. Ingestion of inhibitor with prodrug provides for a decreaseddrug Cmax relative to the drug Cmax that results from ingestion of thesame amount of prodrug in the absence of inhibitor. Panel A alsoillustrates that the total drug exposure following ingestion of prodrugwith inhibitor is also decreased relative to ingestion of the sameamount of prodrug without inhibitor.

Panel B of FIG. 2 provides another example of a dose unit having amodified concentration-time PK profile. As in Panel A, the solid topline represents drug concentration over time in blood or plasmafollowing ingestion of prodrug without inhibitor, while the dashed lowerline represents drug concentration following ingestion of the sameamount of prodrug with inhibitor. In this example, the dose unitprovides a PK profile having a decreased drug Cmax, decreased drugexposure, and a delayed drug Tmax (i.e., decreased (1/drug Tmax)relative to ingestion of the same dose of prodrug without inhibitor.

Panel C of FIG. 2 provides another example of a dose unit having amodified concentration-time PK profile. As in Panel A, the solid linerepresents drug concentration over time in blood or plasma followingingestion of prodrug without inhibitor, while the dashed line representsdrug concentration following ingestion of the same amount of prodrugwith inhibitor. In this example, the dose unit provides a PK profilehaving a delayed drug Tmax (i.e., decreased (1/drug Tmax) relative toingestion of the same dose of prodrug without inhibitor.

Dose units that provide for a modified PK profile (e.g., a decreaseddrug Cmax and/or delayed drug Tmax as compared to, a PK profile of drugor a PK profile of prodrug without inhibitor), find use in tailoring ofdrug dose according to a patient's needs (e.g., through selection of aparticular dose unit and/or selection of a dosage regimen), reduction ofside effects, and/or improvement in patient compliance (as compared toside effects or patient compliance associated with drug or with prodrugwithout inhibitor). As used herein, “patient compliance” refers towhether a patient follows the direction of a clinician (e.g., aphysician) including ingestion of a dose that is neither significantlyabove nor significantly below that prescribed. Such dose units alsoreduce the risk of misuse, abuse or overdose by a patient as compared tosuch risk(s) associated with drug or prodrug without inhibitor. Forexample, dose units with a decreased drug Cmax provide less reward foringestion than does a dose of the same amount of drug, and/or the sameamount of prodrug without inhibitor.

Dose Units Providing Modified PK Profiles Upon Ingestion of MultipleDose Units

A dose unit of the present disclosure can be adapted to provide for adesired PK profile (e.g., a concentration-time PK profile orconcentration-dose PK profile) following ingestion of multiples of adose unit (e.g., at least 2, at least 3, at least 4, or more doseunits). A concentration-dose PK profile refers to the relationshipbetween a selected PK parameter and a number of single dose unitsingested. Such a profile can be dose proportional, linear (a linear PKprofile) or nonlinear (a nonlinear PK profile). A modifiedconcentration-dose PK profile can be provided by adjusting the relativeamounts of prodrug and inhibitor contained in a single dose unit and/orby using a different prodrug and/or inhibitor.

FIG. 3 provides schematics of examples of concentration-dose PK profiles(exemplified by drug Cmax, Y axis) that can be provided by ingestion ofmultiples of a dose unit (X axis) of the present disclosure. Eachprofile can be compared to a concentration-dose PK profile provided byincreasing doses of drug alone, where the amount of drug in the blood orplasma from one dose represents a therapeutically effective amountequivalent to the amount of drug released into the blood or plasma byone dose unit of the disclosure. Such a “drug alone” PK profile istypically dose proportional, having a forty-five degree angle positivelinear slope. It is also to be appreciated that a concentration-dose PKprofile resulting from ingestion of multiples of a dose unit of thedisclosure can also be compared to other references, such as aconcentration-dose PK profile provided by ingestion of an increasingnumber of doses of prodrug without inhibitor wherein the amount of drugreleased into the blood or plasma by a single dose of prodrug in theabsence of inhibitor represents a therapeutically effective amountequivalent to the amount of drug released into the blood or plasma byone dose unit of the disclosure.

As illustrated by the relationship between prodrug and inhibitorconcentration in FIG. 1, a dose unit can include inhibitor in an amountthat does not detectably affect drug release following ingestion.Ingestion of multiples of such a dose unit can provide aconcentration-dose PK profile such that the relationship between numberof dose units ingested and PK parameter value is linear with a positiveslope, which is similar to, for example, a dose proportional PK profileof increasing amounts of prodrug alone. Panel A of FIG. 3 depicts such aprofile. Dose units that provide a concentration-dose PK profile havingsuch an undetectable change in drug Cmax in vivo compared to the profileof prodrug alone can find use in thwarting enzyme conversion of prodrugfrom a dose unit that has sufficient inhibitor to reduce or prevent invitro cleavage of the enzyme-cleavable prodrug by its respective enzyme.

Panel B in FIG. 3 represents a concentration-dose PK profile such thatthe relationship between the number of dose units ingested and a PKparameter value is linear with positive slope, where the profileexhibits a reduced slope relative to panel A. Such a dose unit providesa profile having a decreased PK parameter value (e.g., drug Cmax)relative to a reference PK parameter value exhibiting doseproportionality.

Concentration-dose PK profiles following ingestion of multiples of adose unit can be non-linear. Panel C in FIG. 3 represents an example ofa non-linear, biphasic concentration-dose PK profile. In this example,the biphasic concentration-dose PK profile contains a first phase overwhich the concentration-dose PK profile has a positive rise, and then asecond phase over which the relationship between number of dose unitsingested and a PK parameter value (e.g., drug Cmax) is relatively flat(substantially linear with zero slope). For such a dose unit, forexample, drug Cmax can be increased for a selected number of dose units(e.g., 2, 3, or 4 dose units). However, ingestion of additional doseunits does not provide for a significant increase in drug Cmax.

Panel D in FIG. 3 represents another example of a non-linear, biphasicconcentration-dose PK profile. In this example, the biphasicconcentration-dose PK profile is characterized by a first phase overwhich the concentration-dose PK profile has a positive rise and a secondphase over which the relationship between number of dose units ingestedand a PK parameter value (e.g., drug Cmax) declines. Dose units thatprovide this concentration-dose PK profile provide for an increase indrug Cmax for a selected number of ingested dose units (e.g., 2, 3, or 4dose units). However, ingestion of further additional dose units doesnot provide for a significant increase in drug Cmax and instead providesfor decreased drug Cmax.

Panel E in FIG. 3 represents a concentration-dose PK profile in whichthe relationship between the number of dose units ingested and a PKparameter (e.g., drug Cmax) is linear with zero slope. Such dose unitsdo not provide for a significant increase or decrease in drug Cmax withingestion of multiples of dose units.

Panel F in FIG. 3 represents a concentration-dose PK profile in whichthe relationship between number of dose units ingested and a PKparameter value (e.g., drug Cmax) is linear with a negative slope. Thusdrug Cmax decreases as the number of dose units ingested increases.

Dose units that provide for concentration-dose PK profiles whenmultiples of a dose unit are ingested find use in tailoring of a dosageregimen to provide a therapeutic level of released drug while reducingthe risk of overdose, misuse, or abuse. Such reduction in risk can becompared to a reference, e.g., to administration of drug alone orprodrug alone. In one embodiment, risk is reduced compared toadministration of a drug or prodrug that provides a proportionalconcentration-dose PK profile. A dose unit that provides for aconcentration-dose PK profile can reduce the risk of patient overdosethrough inadvertent ingestion of dose units above a prescribed dosage.Such a dose unit can reduce the risk of patient misuse (e.g., throughself-medication). Such a dose unit can discourage abuse throughdeliberate ingestion of multiple dose units. For example, a dose unitthat provides for a biphasic concentration-dose PK profile can allow foran increase in drug release for a limited number of dose units ingested,after which an increase in drug release with ingestion of more doseunits is not realized. In another example, a dose unit that provides fora concentration-dose PK profile of zero slope can allow for retention ofa similar drug release profile regardless of the number of dose unitsingested.

Ingestion of multiples of a dose unit can provide for adjustment of a PKparameter value relative to that of ingestion of multiples of the samedose (either as drug alone or as a prodrug) in the absence of inhibitorsuch that, for example, ingestion of a selected number (e.g., 2, 3, 4 ormore) of a single dose unit provides for a decrease in a PK parametervalue compared to ingestion of the same number of doses in the absenceof inhibitor.

Pharmaceutical compositions include those having an inhibitor to providefor protection of a therapeutic compound from degradation in the GItract. Inhibitor can be combined with a drug (i.e., not a prodrug) toprovide for protection of the drug from degradation in the GI system. Inthis example, the composition of inhibitor and drug provide for amodified PK profile by increasing a PK parameter. Inhibitor can also becombined with a prodrug that is susceptible to degradation by a GIenzyme and has a site of action outside the GI tract. In thiscomposition, the inhibitor protects ingested prodrug in the GI tractprior to its distribution outside the GI tract and cleavage at a desiredsite of action.

Methods Used to Define Relative Amounts of Prodrug and Inhibitor in aDose Unit

Dose units that provide for a desired PK profile, such as a desiredconcentration-time PK profile and/or a desired concentration-dose PKprofile, can be made by combining a prodrug and an inhibitor in a doseunit in relative amounts effective to provide for release of drug thatprovides for a desired drug PK profile following ingestion by a patient.

Prodrugs can be selected as suitable for use in a dose unit bydetermining the trypsin-mediated drug release competency of the prodrug.This can be accomplished in vitro, in vivo or ex vivo.

In vitro assays can be conducted by combining a prodrug with trypsin ina reaction mixture. Trypsin can be provided in the reaction mixture inan amount sufficient to catalyze cleavage of the prodrug. Assays areconducted under suitable conditions, and optionally may be underconditions that mimic those found in a GI tract of a subject, e.g.,human. “Prodrug conversion” refers to release of drug from prodrug.Prodrug conversion can be assessed by detecting a level of a product ofprodrug conversion (e.g., released drug) and/or by detecting a level ofprodrug that is maintained in the presence of trypsin. Prodrugconversion can also be assessed by detecting the rate at which a productof prodrug conversion occurs or the rate at which prodrug disappears. Anincrease in released drug, or a decrease in prodrug, indicate prodrugconversion has occurred. Prodrugs that exhibit an acceptable level ofprodrug conversion in the presence of trypsin within an acceptableperiod of time are suitable for use in a dose unit in combination with atrypsin inhibitor.

In vivo assays can assess the suitability of a prodrug for use in a doseunit by administration of the prodrug to an animal (e.g., a human ornon-human animal, e.g., rat, dog, pig, etc.). Such administration can beenteral (e.g., oral administration). Prodrug conversion can be detectedby, for example, detecting a product of prodrug conversion (e.g.,released drug or a metabolite of released drug) or detecting prodrug inblood or plasma of the animal at a desired time point(s) followingadministration.

Ex vivo assays, such as a gut loop or inverted gut loop assay, canassess the suitability of a prodrug for use in a dose unit by, forexample, administration of the prodrug to a ligated section of theintestine of an animal. Prodrug conversion can be detected by, forexample, detecting a product of prodrug conversion (e.g., released drugor a metabolite of released drug) or detecting prodrug in the ligatedgut loop of the animal at a desired time point(s) followingadministration.

Inhibitors are generally selected based on, for example, activity ininteracting with trypsin that mediates release of drug from a prodrugwith which the inhibitor is to be co-dosed. Such assays can be conductedin the presence of enzyme either with or without prodrug. Inhibitors canalso be selected according to properties such as half-life in the GIsystem, potency, avidity, affinity, molecular size and/or enzymeinhibition profile (e.g., steepness of inhibition curve in an enzymeactivity assay, inhibition initiation rate). Inhibitors for use inprodrug-inhibitor combinations can be selected through use of in vitro,in vivo and/or ex vivo assays.

One embodiment is a method for identifying a prodrug and a trypsininhibitor suitable for formulation in a dose unit wherein the methodcomprises combining a prodrug (e.g., Compound KC-8), a trypsininhibitor, and trypsin in a reaction mixture and detecting prodrugconversion. Such a combination is tested for an interaction between theprodrug, inhibitor and enzyme, i.e., tested to determine how theinhibitor will interact with the enzyme that mediatesenzymatically-controlled release of the drug from the prodrug. In oneembodiment, a decrease in prodrug conversion in the presence of thetrypsin inhibitor as compared to prodrug conversion in the absence ofthe trypsin inhibitor indicates the prodrug and trypsin inhibitor aresuitable for formulation in a dose unit. Such a method can be an invitro assay.

One embodiment is a method for identifying a prodrug and a trypsininhibitor suitable for formulation in a dose unit wherein the methodcomprises administering to an animal a prodrug (e.g., Compound KC-8) anda trypsin inhibitor and detecting prodrug conversion. In one embodiment,a decrease in prodrug conversion in the presence of the trypsininhibitor as compared to prodrug conversion in the absence of thetrypsin inhibitor indicates the prodrug and trypsin inhibitor aresuitable for formulation in a dose unit. Such a method can be an in vivoassay; for example, the prodrug and trypsin inhibitor can beadministered orally. Such a method can also be an ex vivo assay; forexample, the prodrug and trypsin inhibitor can be administered orally orto a tissue, such as an intestine, that is at least temporarily exposed.Detection can occur in the blood or plasma or respective tissue. As usedherein, tissue refers to the tissue itself and can also refer tocontents within the tissue.

One embodiment is a method for identifying a prodrug and a trypsininhibitor suitable for formulation in a dose unit wherein the methodcomprises administering a prodrug and a trypsin inhibitor to an animaltissue that has removed from an animal and detecting prodrug conversion.In one embodiment, a decrease in prodrug conversion in the presence ofthe trypsin inhibitor as compared to prodrug conversion in the absenceof the trypsin inhibitor indicates the prodrug and trypsin inhibitor aresuitable for formulation in a dose unit.

In vitro assays can be conducted by combining a prodrug, a trypsininhibitor and trypsin in a reaction mixture. Trypsin can be provided inthe reaction mixture in an amount sufficient to catalyze cleavage of theprodrug, and assays conducted under suitable conditions, optionallyunder conditions that mimic those found in a GI tract of a subject,e.g., human. Prodrug conversion can be assessed by detecting a level ofa product of prodrug conversion (e.g., released drug) and/or bydetecting a level of prodrug maintained in the presence of trypsin.Prodrug conversion can also be assessed by detecting the rate at which aproduct of prodrug conversion occurs or the rate at which prodrugdisappears. Prodrug conversion that is modified in the presence ofinhibitor as compared to a level of prodrug conversion in the absence ofinhibitor indicates the inhibitor is suitable for attenuation of prodrugconversion and for use in a dose unit. Reaction mixtures having a fixedamount of prodrug and increasing amounts of inhibitor, or a fixed amountof inhibitor and increasing amounts of prodrug, can be used to identifyrelative amounts of prodrug and inhibitor which provide for a desiredmodification of prodrug conversion.

In vivo assays can assess combinations of prodrugs and inhibitors byco-dosing of prodrug and inhibitor to an animal. Such co-dosing can beenteral. “Co-dosing” refers to administration of prodrug and inhibitoras separate doses or a combined dose (i.e., in the same formulation).Prodrug conversion can be detected by, for example, detecting a productof prodrug conversion (e.g., released drug or drug metabolite) ordetecting prodrug in blood or plasma of the animal at a desired timepoint(s) following administration. Combinations of prodrug and inhibitorcan be identified that provide for a prodrug conversion level thatyields a desired PK profile as compared to, for example, prodrug withoutinhibitor.

Combinations of relative amounts of prodrug and inhibitor that providefor a desired PK profile can be identified by dosing animals with afixed amount of prodrug and increasing amounts of inhibitor, or with afixed amount of inhibitor and increasing amounts of prodrug. One or morePK parameters can then be assessed, e.g., drug Cmax, drug Tmax, and drugexposure. Relative amounts of prodrug and inhibitor that provide for adesired PK profile are identified as amounts of prodrug and inhibitorfor use in a dose unit. The PK profile of the prodrug and inhibitorcombination can be, for example, characterized by a decreased PKparameter value relative to prodrug without inhibitor. A decrease in thePK parameter value of an inhibitor-to-prodrug combination (e.g., adecrease in drug Cmax, a decrease in 1/drug Tmax (i.e., a delay in drugTmax) or a decrease in drug exposure) relative to a corresponding PKparameter value following administration of prodrug without inhibitorcan be indicative of an inhibitor-to-prodrug combination that canprovide a desired PK profile. Assays can be conducted with differentrelative amounts of inhibitor and prodrug.

In vivo assays can be used to identify combinations of prodrug andinhibitor that provide for dose units that provide for a desiredconcentration-dose PK profile following ingestion of multiples of thedose unit (e.g., at least 2, at least 3, at least 4 or more). Ex vivoassays can be conducted by direct administration of prodrug andinhibitor into a tissue and/or its contents of an animal, such as theintestine, including by introduction by injection into the lumen of aligated intestine (e.g., a gut loop, or intestinal loop, assay, or aninverted gut assay). An ex vivo assay can also be conducted by excisinga tissue and/or its contents from an animal and introducing prodrug andinhibitor into such tissues and/or contents.

For example, a dose of prodrug that is desired for a single dose unit isselected (e.g., an amount that provides an efficacious plasma druglevel). A multiple of single dose units for which a relationship betweenthat multiple and a PK parameter to be tested is then selected. Forexample, if a concentration-dose PK profile is to be designed foringestion of 2, 3, 4, 5, 6, 7, 8, 9 or 10 dose units, then the amount ofprodrug equivalent to ingestion of that same number of dose units isdetermined (referred to as the “high dose”). The multiple of dose unitscan be selected based on the number of ingested pills at which drug Cmaxis modified relative to ingestion of the single dose unit. If, forexample, the profile is to provide for abuse deterrence, then a multipleof 10 can be selected, for example. A variety of different inhibitors(e.g., from a panel of inhibitors) can be tested using differentrelative amounts of inhibitor and prodrug. Assays can be used toidentify suitable combination(s) of inhibitor and prodrug to obtain asingle dose unit that is therapeutically effective, wherein such acombination, when ingested as a multiple of dose units, provides amodified PK parameter compared to ingestion of the same multiple of drugor prodrug alone (wherein a single dose of either drug or prodrug alonereleases into blood or plasma the same amount of drug as is released bya single dose unit).

Increasing amounts of inhibitor are then co-dosed to animals with thehigh dose of prodrug. The dose level of inhibitor that provides adesired drug Cmax following ingestion of the high dose of prodrug isidentified and the resultant inhibitor-to-prodrug ratio determined.

Prodrug and inhibitor are then co-dosed in amounts equivalent to theinhibitor-to-prodrug ratio that provided the desired result at the highdose of prodrug. The PK parameter value of interest (e.g., drug Cmax) isthen assessed. If a desired PK parameter value results followingingestion of the single dose unit equivalent, then single dose unitsthat provide for a desired concentration-dose PK profile are identified.For example, where a zero dose linear profile is desired, the drug Cmaxfollowing ingestion of a single dose unit does not increasesignificantly following ingestion of a multiple number of the singledose units.

Methods for Manufacturing, Formulating, and Packaging Dose Units

Dose units of the present disclosure can be made using manufacturingmethods available in the art and can be of a variety of forms suitablefor enteral (including oral, buccal and sublingual) administration, forexample as a tablet, capsule, thin film, powder, suspension, solution,syrup, dispersion or emulsion. The dose unit can contain componentsconventional in pharmaceutical preparations, e.g. one or more carriers,binders, lubricants, excipients (e.g., to impart controlled releasecharacteristics), pH modifiers, flavoring agents (e.g., sweeteners),bulking agents, coloring agents or further active agents. Dose units ofthe present disclosure can include can include an enteric coating orother component(s) to facilitate protection from stomach acid, wheredesired.

Dose units can be of any suitable size or shape. The dose unit can be ofany shape suitable for enteral administration, e.g., ellipsoid,lenticular, circular, rectangular, cylindrical, and the like.

Dose units provided as dry dose units can have a total weight of fromabout 1 microgram to about 1 gram, and can be from about 5 micrograms to1.5 grams, from about 50 micrograms to 1 gram, from about 100 microgramsto 1 gram, from 50 micrograms to 750 milligrams, and may be from about 1microgram to 2 grams.

Dose units can comprise components in any relative amounts. For example,dose units can be from about 0.1% to 99% by weight of active ingredients(i.e., prodrug and inhibitor) per total weight of dose unit (0.1% to 99%total combined weight of prodrug and inhibitor per total weight ofsingle dose unit). In some embodiments, dose units can be from 10% to50%, from 20% to 40%, or about 30% by weight of active ingredients pertotal weight dose unit.

Dose units can be provided in a variety of different forms andoptionally provided in a manner suitable for storage. For example, doseunits can be disposed within a container suitable for containing apharmaceutical composition. The container can be, for example, a bottle(e.g., with a closure device, such as a cap), a blister pack (e.g.,which can provide for enclosure of one or more dose units per blister),a vial, flexible packaging (e.g., sealed Mylar or plastic bags), anampule (for single dose units in solution), a dropper, thin film, a tubeand the like.

Containers can include a cap (e.g., screw cap) that is removablyconnected to the container over an opening through which the dose unitsdisposed within the container can be accessed.

Containers can include a seal which can serve as a tamper-evident and/ortamper-resistant element, which seal is disrupted upon access to a doseunit disposed within the container. Such seal elements can be, forexample, a frangible element that is broken or otherwise modified uponaccess to a dose unit disposed within the container. Examples of suchfrangible seal elements include a seal positioned over a containeropening such that access to a dose unit within the container requiresdisruption of the seal (e.g., by peeling and/or piercing the seal).Examples of frangible seal elements include a frangible ring disposedaround a container opening and in connection with a cap such that thering is broken upon opening of the cap to access the dose units in thecontainer.

Dry and liquid dose units can be placed in a container (e.g., bottle orpackage, e.g., a flexible bag) of a size and configuration adapted tomaintain stability of dose units over a period during which the doseunits are dispensed into a prescription. For example, containers can besized and configured to contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100or more single dry or liquid dose units. The containers can be sealed orresealable. The containers can packaged in a carton (e.g., for shipmentfrom a manufacturer to a pharmacy or other dispensary). Such cartons canbe boxes, tubes, or of other configuration, and may be made of anymaterial (e.g., cardboard, plastic, and the like). The packaging systemand/or containers disposed therein can have one or more affixed labels(e.g., to provide information such as lot number, dose unit type,manufacturer, and the like).

The container can include a moisture barrier and/or light barrier, e.g.,to facilitate maintenance of stability of the active ingredients in thedose units contained therein. Where the dose unit is a dry dose unit,the container can include a desiccant pack which is disposed within thecontainer. The container can be adapted to contain a single dose unit ormultiples of a dose unit. The container can include a dispensing controlmechanism, such as a lock out mechanism that facilitates maintenance ofdosing regimen.

The dose units can be provided in solid or semi-solid form, and can be adry dose unit. “Dry dose unit” refers to a dose unit that is in otherthan in a completely liquid form. Examples of dry dose units include,for example, tablets, capsules (e.g., solid capsules, capsulescontaining liquid), thin film, microparticles, granules, powder and thelike. Dose units can be provided as liquid dose units, where the doseunits can be provided as single or multiple doses of a formulationcontaining prodrug and inhibitor in liquid form. Single doses of a dryor liquid dose unit can be disposed within a sealed container, andsealed containers optionally provided in a packaging system, e.g., toprovide for a prescribed number of doses, to provide for shipment ofdose units, and the like.

Dose units can be formulated such that the prodrug and inhibitor arepresent in the same carrier, e.g., solubilized or suspended within thesame matrix. Alternatively, dose units can be composed of two or moreportions, where the prodrug and inhibitor can be provided in the same ordifferent portions, and can be provided in adjacent or non-adjacentportions.

Dose units can be provided in a container in which they are disposed,and may be provided as part of a packaging system (optionally withinstructions for use). For example, dose units containing differentamounts of prodrug can be provided in separate containers, whichcontainers can be disposed with in a larger container (e.g., tofacilitate protection of dose units for shipment). For example, one ormore dose units as described herein can be provided in separatecontainers, where dose units of different composition are provided inseparate containers, and the separate containers disposed within packagefor dispensing.

In another example, dose units can be provided in a double-chambereddispenser where a first chamber contains a prodrug formulation and asecond chamber contains an inhibitor formulation. The dispenser can beadapted to provide for mixing of a prodrug formulation and an inhibitorformulation prior to ingestion. For example, the two chambers of thedispenser can be separated by a removable wall (e.g., frangible wall)that is broken or removed prior to administration to allow mixing of theformulations of the two chambers. The first and second chambers canterminate into a dispensing outlet, optionally through a common chamber.The formulations can be provided in dry or liquid form, or a combinationthereof. For example, the formulation in the first chamber can be liquidand the formulation in the second chamber can be dry, both can be dry,or both can be liquid.

Dose units that provide for controlled release of prodrug, of inhibitor,or of both prodrug and inhibitor are contemplated by the presentdisclosure, where “controlled release” refers to release of one or bothof prodrug and inhibitor from the dose unit over a selected period oftime and/or in a pre-selected manner.

Methods of Use of Dose Units

Dose units are advantageous because they find use in methods to reduceside effects and/or improve tolerability of drugs to patients in needthereof by, for example, limiting a PK parameter as disclosed herein.The present disclosure thus provides methods to reduce side effects byadministering a dose unit of the present disclosure to a patient in needso as to provide for a reduction of side effects as compared to thoseassociated with administration of drug and/or as compared toadministration of prodrug without inhibitor. The present disclosure alsoprovides methods to improve tolerability of drugs by administering adose unit of the present disclosure to a patient in need so as toprovide for improvement in tolerability as compared to administration ofdrug and/or as compared to administration of prodrug without inhibitor.

Dose units find use in methods for increasing patient compliance of apatient with a therapy prescribed by a clinician, where such methodsinvolve directing administration of a dose unit described herein to apatient in need of therapy so as to provide for increased patientcompliance as compared to a therapy involving administration of drugand/or as compared to administrations of prodrug without inhibitor. Suchmethods can help increase the likelihood that a clinician-specifiedtherapy occurs as prescribed.

Dose units can provide for enhanced patient compliance and cliniciancontrol. For example, by limiting a PK parameter (e.g., such as drugCmax or drug exposure) when multiples (e.g., two or more, three or more,or four or more) dose units are ingested, a patient requiring a higherdose of drug must seek the assistance of a clinician. The dose units canprovide for control of the degree to which a patient can readily“self-medicate”, and further can provide for the patient to adjust doseto a dose within a permissible range. Dose units can provide for reducedside effects, by for example, providing for delivery of drug at anefficacious dose but with a modified PK profile over a period oftreatment, e.g., as defined by a decreased PK parameter (e.g., decreaseddrug Cmax, decreased drug exposure).

Dose units find use in methods to reduce the risk of unintended overdoseof drug that can follow ingestion of multiple doses taken at the sametime or over a short period of time. Such methods of the presentdisclosure can provide for reduction of risk of unintended overdose ascompared to risk of unintended overdose of drug and/or as compared torisk of unintended overdose of prodrug without inhibitor. Such methodsinvolve directing administration of a dosage described herein to apatient in need of drug released by conversion of the prodrug. Suchmethods can help avoid unintended overdosing due to intentional orunintentional misuse of the dose unit.

The present disclosure provides methods to reduce misuse and abuse of adrug, as well as to reduce risk of overdose, that can accompanyingestion of multiples of doses of a drug, e.g., ingested at the sametime. Such methods generally involve combining in a dose unit a prodrugand a trypsin inhibitor that mediates release of drug from the prodrug,where the inhibitor is present in the dose unit in an amount effectiveto attenuate release of drug from the prodrug, e.g., following ingestionof multiples of dose units by a patient. Such methods provide for amodified concentration-dose PK profile while providing therapeuticallyeffective levels from a single dose unit, as directed by the prescribingclinician. Such methods can provide for, for example, reduction of risksthat can accompany misuse and/or abuse of a prodrug, particularly whereconversion of the prodrug provides for release of a narcotic or otherdrug of abuse (e.g., opioid). For example, when the prodrug provides forrelease of a drug of abuse, dose units can provide for reduction ofreward that can follow ingestion of multiples of dose units of a drug ofabuse.

Dose units can provide clinicians with enhanced flexibility inprescribing drug. For example, a clinician can prescribe a dosageregimen involving different dose strengths, which can involve two ormore different dose units of prodrug and inhibitor having differentrelative amounts of prodrug, different amounts of inhibitor, ordifferent amounts of both prodrug and inhibitor. Such different strengthdose units can provide for delivery of drug according to different PKparameters (e.g., drug exposure, drug Cmax, and the like as describedherein). For example, a first dose unit can provide for delivery of afirst dose of drug following ingestion, and a second dose unit canprovide for delivery of a second dose of drug following ingestion. Thefirst and second prodrug doses of the dose units can be differentstrengths, e.g., the second dose can be greater than the first dose. Aclinician can thus prescribe a collection of two or more, or three ormore dose units of different strengths, which can be accompanied byinstructions to facilitate a degree of self-medication, e.g., toincrease delivery of an opioid drug according to a patient's needs totreat pain.

Thwarting Tampering by Trypsin Mediated Release of Oxycodone fromProdrug

The disclosure provides for a composition comprising Compound KC-8 and atrypsin inhibitor that reduces drug abuse potential. A trypsin inhibitorcan thwart the ability of a user to apply trypsin to effect the releaseof oxycodone from the ketone-modified oxycodone prodrug, Compound KC-8,in vitro. For example, if an abuser attempts to incubate trypsin with acomposition of the embodiments that includes Compound KC-8 and a trypsininhibitor, the trypsin inhibitor can reduce the action of the addedtrypsin, thereby thwarting attempts to release oxycodone for purposes ofabuse.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the embodiments, and are not intended to limit the scope ofwhat the inventors regard as their invention nor are they intended torepresent that the experiments below are all or the only experimentsperformed. Efforts have been made to ensure accuracy with respect tonumbers used (e.g. amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is weight averagemolecular weight, temperature is in degrees Celsius, and pressure is ator near atmospheric. Standard abbreviations may be used.

Synthesis of Ketone-modified Opioid Prodrugs Example 1 Synthesis ofoxycodone 6-(N-methyl-N-(2-amino)ethylcarbamate (Compound KC-19)

Preparation of Compound A

2-(Aminoethyl)-methyl-carbamic acid benzyl ester (2.0 g, 9.6 mmol) wasdissolved in dichloroethene (DCE) (20 mL) at room temperature. Triethylamine (NEt₃) (1.40 mL, 11.5 mmol) was added, followed by di-tert-butyldicarbonate (BOC₂O) (10.5 g, 48 mmol) and dimethylaminopyridine (DMAP)(120 mg). The reaction mixture was stirred at room temperature undernitrogen (N₂) for 2 h and then heated at 60° C. for 16 h. The reactionmixture was then concentrated. The residue was purified by silica gelchromatography, using 4/1 hexanes/EtOAc, to give Compound A in 86% yield(3.4 g, 8.3 mmol). MS: (m/z) calc: 408.2, observed (M+Na⁺) 431.9.

Preparation of Compound B

Compound A (1.3 g, 3.18 mmol) was dissolved in methanol/EtOAc (10 mL/3mL respectively). The mixture was degassed and saturated with N₂.Palladium on carbon (Pd/C) (330 mg, 5% on carbon) was added. The mixturewas shaken in a Parr hydrogenator flask (50 psi H₂) for 4 h. The mixturewas then filtered through a celite pad, and the filtrate wasconcentrated to give Compound B (1.08 g, yield exceeded quantitative).Compound B was used without further purification.

Preparation of Compound C

Compound B (500 mg, 1.82 mmol) and NEt₃ (0.4 mL, 2.74 mmol) were mixedtogether in dichloromethane (4 mL). The mixture was added to apre-chilled 0° C. solution of phosgene (5.5 mL, 0.5 M in toluene). Thereaction mixture was stirred at 0° C. for 1 h, followed by dilution withether (20 mL) and filtration through filter paper. The filtrate wasconcentrated and passed through a short silica gel column (10 cm×3 cm),and eluted with 3/1 hexanes/EtOAc. The fractions were concentrated togive N,N-Bis(tert-butyl)N′-2-(chlorocarbonyl(methyl)amino)ethylcarbamate(Compound C) as a colorless solid in quantitative yield (615 mg, 1.82mmol). MS: (m/z) calc: 336.1, observed (M+Na⁺) 359.8.

Synthesis of Oxycodone 6-(N-methyl-N-(2-amino)ethylcarbamate (CompoundKC-19)

Oxycodone free base (6.5 g, 20.6 mmol) was dissolved in dry, degassedtetrahydrofuran (120 mL), and the mixture was cooled to −10° C. using adry ice/acetone bath. Potassium bis(trimethylsilyl)amide (KHMDS) (103.0mL, 51.6 mmol, 0.5 M in toluene) was added via cannula. The mixture wasstirred under N₂ at below −5° C. for 30 min.N,N-Bis(tert-butyl)N′-2-(chlorocarbonyl(methyl)amino)ethylcarbamate (8.0g, 23.7 mmol), (Compound C) in THF (30 mL) was then added via cannulaover 15 min. The mixture was stirred at −5° C. for 30 min. Anotherportion of carbamoyl chloride (4.0 g, 11.9 mmol) in THF (10 mL) wasadded. The reaction was stirred at room temperature for 2 h. Sodiumbicarbonate (10 mL, sat. aq.) was added. The mixture was concentrated invacuo to half of its initial volume. EtOAc (50 mL) was added, and layerswere separated. The organic phase was further washed with water (3×20mL) and brine (40 mL), and then was concentrated. The residue waspurified by silica gel chromatography, using DCM/MeOH (gradient 100/1 to100/15) to afford a white foam in 55% yield (7.0 g, 13.4 mmol). Thismaterial was dissolved in a 1:1 mixture of DCM/trifluoroacetic acid(TFA) (20 mL/20 mL) at room temperature and stirred for 1 h. Thesolution was then concentrated in vacuo to afford a TFA salt ofoxycodone 6-(N-methyl-N-(2-amino)ethylcarbamate (Compound KC-19) as athick oil (7.3 g, 11.4 mmol, 99% purity). MS: (m/z) calc: 415.2,observed (M+H⁺) 416.5.

Example 2 Synthesis ofN-1-[2-(oxycodone-6-enol-carbonyl-methyl-amino)-ethylamine]-arginine-malonicacid (Compound KC-3) [also named:N-{(S)-4-guanidino-1-[2-(methyl-[(5R,9R,13S,14S)-4,5a-epoxy-6,7-didehydro-14-hydroxy-3-methoxy-[7-methylmorphinan-6-oxy]carbonyl-amino)-ethylcarbamoyl]-butyl}-malonicacid]

Preparation of Compound D

A solution of N-methylethylenediamine (27.0 g, 364 mmol) and ethyltrifluoroacetate (96.6 mL, 812 mmol) in a mixture of ACN (350 mL) andwater (7.8 mL, 436 mmol) was refluxed with stifling overnight. Solventswere evaporated in vacuo. The residue was re-evaporated with i-PrOH(3×100 mL), followed by heat-cool crystallization from DCM (500 mL).Formed crystals were filtered, washed with DCM and dried in vacuo toprovide Compound D (88.3 g, 85%) as white solid powder.

Preparation of Compound E

A solution of Compound D (88.2 g, 311 mmol) and DIEA (54.1 mL, 311 mmol)in THF (350 mL) was cooled in an ice bath, followed by the addition of asolution of N-(benzyloxycarbonyl)succinimide (76.6 g, 307 mmol) in THF(150 mL) dropwise over the period of 20 min. The temperature of thereaction mixture was raised to ambient temperature and stirring wascontinued for an additional 30 min. Solvents were then evaporated andthe resulting residue was dissolved in EtOAc (600 mL). The organic layerwas extracted with 5% aq. NaHCO₃ (2×150 mL) and brine (150 mL). Theorganic layer was evaporated to provide Compound E as yellowish oil.LC-MS [M+H] 305.1 (C₁₃H₁₅F₃N₂O₃+H, calc: 305.3). Compound E was useddirectly in the next reaction without purification as a MeOH solution.

Preparation of Compound F

To a solution of Compound E (˜311 mmol) in MeOH (1.2 L) was added asolution of LiOH (14.9 g, 622 mmol) in water (120 mL). The reactionmixture was stirred at ambient temperature for 3 h. Solvents wereevaporated to 75% of the initial volume followed by dilution with water(400 mL). The solution was extracted with EtOAc (2×300 mL). The organiclayer was washed with brine (200 mL), dried over MgSO₄ and evaporated invacuo. The residue was dissolved in ether (300 mL) and treated with 2 NHCl/ether (200 mL). Formed precipitate was filtrated, washed with etherand dried in vacuo to provide the hydrochloric salt of Compound F (67.8g, 89%) as a white solid. LC-MS [M+H] 209.0 (C₁₁H₁₆N₂O₂+H, calc: 209.3).Compound F was used directly in the next reaction without purificationas a DMF solution.

Preparation of Compound G

A solution of Boc-Arg(Pbf)-OH (16.0 g, ˜30.4 mmol), Compound Fhydrochloride (8.2 g, 33.4 mmol), and DIEA (16.9 mL, 97.2 mmol) in DMF(150 mL) was cooled in an ice bath followed by the addition of asolution of HATU (13.8 g, 36.4 mmol) dropwise over 20 min. Thetemperature of the reaction mixture was raised to ambient temperature,and stirring was continued for an additional 1 h. The reaction mixturewas diluted with EtOAc (1 L) and extracted with water (3×200 mL) andbrine (200 mL). The organic layer was dried over MgSO₄ and evaporated toprovide Compound G (24.4 g, yield exceeded quantitative) as a yellowishoil. LC-MS [M+H] 717.4 (C₃₅H₅₂N₆O₈S+H, calc: 717.9). Compound G was useddirectly in the next reaction without purification as a dioxanesolution.

Preparation of Compound H

Compound G (24.4 g, ˜30.4 mmol) was dissolved in dioxane (150 mL) andtreated with 4 N HCl/dioxane (150 mL, 600 mmol) at ambient temperaturefor 1 h. The solvent was then evaporated. The residue was suspended ini-PrOH (100 mL), and the mixture was evaporated (procedure was repeatedtwice). The residue was then dried in vacuo to provide Compound H (21.1g, yield exceeded quantitative) as a yellowish solid. LC-MS [M+H] 617.5(C₃₀H₄₄N₆O₆S+H, calc: 617.8). Compound H was used directly in the nextreaction without purification as a DMF solution.

Preparation of Compound I

A solution of Compound H (21.1 g, ˜30.4 mmol), mono-tert-butyl malonate(5.9 mL, 36.7 mmol), BOP (16.2 g, 36.7 mmol) and DIEA (14.9 mL, 83.5mmol) in DMF (100 mL) was maintained at ambient temperature for 1 h. Thereaction mixture was diluted with EtOAc (1 L) and extracted with water(500 mL), 5% aq. NaHCO₃ (500 mL), water (3×500 mL) and brine (500 mL).The organic layer was dried over MgSO₄, filtered, and then evaporated toprovide Compound I (24.5 g, 97%) as a yellowish amorphous solid. LC-MS[M+H] 759.6 (C₃₇H₅₄N₆O₉S+H, calc: 759.9). Compound I was used withoutfurther purification.

Preparation of Compound J

Compound I (12.3 g, 16.7 mmol) was dissolved in methanol (100 mL)followed by the addition of a Pd/C (5% wt, 2.0 g) suspension in water (2mL). The reaction mixture was subjected to hydrogenation (Parrapparatus, 70 psi H₂) at ambient temperature for 1 h. The catalyst wasthen filtered and washed with methanol. The filtrate was evaporated invacuo to provide Compound J (10.0 g, 99%) as a colorless amorphoussolid. LC-MS [M+H] 625.5 (C₂₉H₄₈N₆O₇S+H, calc: 625.8). Compound J wasused without further purification.

Preparation of Oxycodone Free Base

Oxycodone hydrochloride (10.0 g, 28.5 mmol) was dissolved in chloroform(150 mL) and washed with 5% aq. NaHCO₃ (50 mL). The organic layer wasdried over MgSO₄ and evaporated. The residue was dried in vacuoovernight to provide oxycodone free base (8.3 g, 93%) as a white solid.

Preparation of Compound K

A solution of oxycodone free base (6.6 g, 21.0 mmol) in THF (400 mL) wascooled to −20° C., followed by addition of a 0.5 M solution of KHMDS intoluene (46.3 mL, 23.1 mmol). The obtained solution was then added to asolution of 4-nitro-phenyl chloroformate (4.3 g, 21.0 mmol) in THF (100mL) dropwise over the period of 20 min at −20° C. The reaction wasmaintained at −20° C. for an additional 1 h, followed by addition of asolution of Compound J (10.0 g, 16.1 mmol) in THF (200 mL) at −20° C.The reaction mixture was allowed to warm to ambient temperature andstirred overnight. Solvents were evaporated in vacuo. The resultingresidue was dissolved in EtOAc (20 mL) and precipitated with ether (1L). The formed precipitate was filtrated, washed with ether and dried invacuo to provide Compound K (13.6 g, 87%) as an off-white solid. LC-MS[M+H] 966.9 (C₄₈H₆₇N₇O₁₂S+H, calc: 966.2).

Synthesis ofN-1-[2-(oxycodone-6-enol-carbonyl-methyl-amino)-ethylamine]-arginine-malonicacid (Compound KC-3)

Compound K (13.6 g, 14.1 mmol) was dissolved in a mixture of 5%m-cresol/TFA (100 mL). The reaction mixture was maintained at ambienttemperature for 1 h, followed by dilution with ethyl ether (1 L). Theformed precipitate was filtered, washed with ether and hexane, and driedin vacuo to provide a TFA salt of Compound KC-3 (11.4 g, 81%) as anoff-white solid. LC-MS [M+H] 658.6 (C₃₁H₄₃N₇O₉+H, calc: 658.7).

The TFA salt of crude Compound KC-3 (11.4 g, 11.4 mmol) was dissolved inwater (50 mL). The obtained solution was subjected to HPLC purification.[Nanosyn-Pack YMC-GEL-ODS A (100-10) C-18 column (75×500 mm); flow rate:250 mL/min; injection volume 50 mL; mobile phase A: 100% water, 0.1%TFA; mobile phase B: 100% ACN, 0.1% TFA; isocratic elution at 0% B in 4min, gradient elution from 0% to 10% B in 20 min, isocratic elution at10% B in 30 min, gradient elution from 10% B to 30% B in 41 min;detection at 254 nm]. Fractions containing Compound KC-3 were combinedand concentrated in vacuo. The TFA counterion of the latter was replacedwith an HCl counterion via lyophilization using 0.1N HCl to provide aHCl salt of Compound KC-3 (4.2 g, 41% yield) as a white solid. LC-MS[M+H] 658.6 (C₃₁H₄₃N₇O₉+H, calc: 658.7).

Example 3 Synthesis ofN-1-[3-(oxycodone-6-enol-carbonyl-methyl-amino)-2,2-dimethyl-propylamine](Compound KC-22) andN-1-[3-(oxycodone-6-enol-carbonyl-methyl-amino)-2,2-dimethyl-propylamine]-arginine-glycine-malonicacid (Compound KC-8)

Preparation of Compound M

A solution of 2,2-dimethyl-1,3-diamino propane (Compound L) (48.0 g,470.6 mmol) in THF (1.0 L) was cooled in an ice bath. Ethyltrifluoroacetate (56 mL, 471 mmol) was added over 30 min via syringe.The mixture was allowed to warm up to ambient temperature, and stiflingwas continued for 14 h. The mixture was then concentrated in vacuo tohalf of its original volume to give crude Compound M as a THF solution,which was used without further purification in the next reaction. LC-MS[M+H] 199.6 (C₇H₁₃F₃N₂O+H, calc: 199.1).

Preparation of Compound N

To a crude solution of Compound M (from previous step) in THF (500 mL)and chilled in an ice bath was added (Boc)₂O in small portions over 15min. The mixture was stirred at ambient temperature for 15 h. Thereaction was then concentrated in vacuo to give intermediate Compound Nin 84% yield (over two steps) (120.0 g, 402.4 mmol) as a sticky oil.LC-MS [M+H] 299.2 (C₁₂H₂₁F₃N₂O₃+H, calc: 299.2). Compound N was useddirectly in the next reaction without further purification.

Preparation of Compound O

Compound N (120 g, 403 mmol) was dissolved in CH₃OH (500 mL) and stirredat ambient temperature. NaOH (100 mL, 10 N aq.) was added dropwise. Themixture was then stirred in a pre-heated oil bath at 50° C. for 3 h. Themixture was cooled to ambient temperature and diluted with water (500mL). Solvents were then removed in vacuo. The residue was extracted withCHCl₃ (3×100 mL). The combined CHCl₃ solution was dried over Na₂SO₄,filtered and concentrated in vacuo to afford crude Compound O in 95%yield (77.0 g, 381 mmol). LC-MS [M+H] 203.8 (C₁₀H₂₂N₂O₂+H, calc: 203.2).Compound O was used directly in the next reaction without furtherpurification.

Preparation of Compound P

Compound O (97.0 g, 480 mmol) was dissolved in CH₂Cl₂ (750 mL). To thiswas added K₂CO₃ (75.0 g, 542.6 mmol) in one portion, followed by portionwise addition of 2-nosyl chloride (108.0 g, 487.3 mmol). The reactionmixture was stirred at ambient temperature for 15 h. Water (200 mL) wasthen added, and the layers were separated. The aqueous layer was againextracted with CH₂Cl₂. The combined CH₂Cl₂ solution was dried overNa₂SO₄, filtered and concentrated in vacuo. The residue was purified bysilica gel chromatography using 3/1 Hexanes/EtOAc to give intermediateCompound P in 83% yield (155.0 g, 400.5 mmol) as a white solid. LC-MS[M+H] 388.8 (C₁₆H₂₅N₃O₆S+H, calc: 388.1).

Preparation of Compound R

Compound P (155.0 g, 400.5 mmol) was dissolved in DMF (500 mL) atambient temperature. K₂CO₃ (83.0 g, 600 mmol) was added in one portion.The mixture was then cooled in an ice water bath. MeI (37.0 mL, 593mmol) was added in small portions via syringe over 10 min. The mixturewas then warmed up to ambient temperature, and stifling was continued atthis temperature for another 2 h. The mixture was concentrated in vacuountil ˜50 mL remained. The remaining mixture containing intermediateCompound Q was cooled in an ice water bath. While stirring, thiophenol(100 mL, 978 mmol) was added via a syringe. The resulted mixture wasstirred at ambient temperature for 6 h. Water (500 mL) was added. Themixture was extracted with EtOAc (100 mL, then 2×500 mL). The combinedEtOAc extracts were extracted with 2N HCl (400 mL, then 2×200 mL). TheHCl extracts were pooled and washed with DCM (500 mL). The acidicsolution was then chilled in an ice water bath and basified by adding 10N NaOH until pH ˜13. CHCl₃ (400 mL, then 2×200 mL) was then used toextract the aqueous solution. The combined CHCl₃ solution was dried overNa₂SO₄ and filtered. Evaporation of solvents in vacuo afforded CompoundR in 67% yield (58.0 g, 268.5 mmol) as a slightly yellowish oil. LC-MS[M+H] 217.6 (C₁₁H₂₄N₂O₂+H, calc: 217.2).

Preparation of Compound S

Oxycodone free base (10.0 g, 31.75 mmol) was dissolved in dry THF (150mL) and the mixture was cooled to −70° C. using a dry ice/acetone bath.KHMDS (64.0 mL, 128.0 mmol, 0.5 M in toluene) was added via syringe over15 min. The mixture was stirred under N₂ for an additional 30 min (bathtemperature −70° C.). In a separate flask was added 4-nitrophenylchloroformate (6.4 g, 31.75 mmol) and THF (10 mL). This mixture was alsochilled to −70° C. using a dry ice/acetone bath. The mixture in thefirst flask (containing deprotonated oxycodone) was then transferred viacannula to the second flask (containing 4-nitrophenyl chloroformate).The transfer occurred over ˜30 min, with the temperature of both flasksbeing maintained at −70° C. during the course of the transfer. Theresulted reaction mixture was further stirred at −70° C. for 30 min. Asolution of Compound R (6.9 g, 31.94 mmol) in THF (15 mL) was then addedvia syringe. The mixture was allowed to stir at −70° C. for 30 min, andthen concentrated in vacuo to afford a gel like residue (˜90% solventremoval). The residue was let stand at ambient temperature for 15 h. Itwas then taken into EtOAc (200 mL) and washed with sat. aq. NaHCO₃ (5×50mL), water (3×40 mL) and brine (50 mL). The residue from theconcentrated EtOAc layer was then purified by silica gel chromatography,using 10/1 CH₃Cl/MeOH to give Compound S in 62% yield (11.0 g, 19.7mmol). LC-MS [M+H] 559.1 (C₃₀H₄₃N₃O₇+H, calc: 558.3).

Preparation ofN-1-[3-(oxycodone-6-enol-carbonyl-methyl-amino)-2,2-dimethyl-propylamine](Compound KC-22)

A solution of Compound S (11.0 g, 19.7 mmol) was treated with a mixtureof TFA and DCM (30 mL/30 mL) for 2 h at ambient temperature. Solventswere then removed in vacuo until a volume of ˜5 mL remained. Et₂O (250mL) was added to precipitate out the product. The resulting precipitatewas filtered, washed with Et₂O (50 mL) and dried to afford crudeCompound KC-22 in 97% yield (11.0 g, 19.2 mmol, 90% purity) as a whitesolid. LC-MS [M+H] 458.9 (C₂₅H₃₅N₃O₅+H, calc: 458.3). Compound KC-22 wasused directly in the next reaction without further purification.

Preparation of Compound U

A solution of Boc-Arg(Pbf)-OH (9.4 g, 17.8 mmol), Compound KC-22 (11.0g, 19.7 mmol, 90% pure) and NEt₃ (10.0 mL, 71.7 mmol) in DMF (80 mL) wascooled in an ice bath, followed by the addition of HATU (6.8 g, 17.9mmol) in portions over 10 min. The ice bath was then removed, and thereaction mixture was stirred at ambient temperature for an additional 1h. The mixture was diluted with EtOAc (150 mL) and extracted with water(3×50 mL) and brine (50 mL). The organic layer was dried over Na₂SO₄ andfiltered; removal of solvents in vacuo provided crude Compound U.Compound U was purified by flash chromatography using CH₂Cl₂ and MeOH toafford Compound U in 79% yield (13.7 g, 14.2 mmol) as a foamy solid.LC-MS [M+H] 967.5 (C₄₉H₇₁N₇O₁₁S+H, calc: 966.5).

Preparation of Compound V

A solution of Compound U (13.7 g, 14.2 mmol) was treated with HCl (4.0Msolution in 1,4-dioxane, 40 mL) at ambient temperature for 90 min.Solvents were removed in vacuo, and the residue was treated with Et₂O(100 mL). The resulting precipitate was filtered off, washed with Et₂O(2×25 mL), and dried to afford crude Compound V in 91% yield (12.1 g,12.9 mmol) as a white solid. LC-MS [M+H] 867.8 (C₄₄H₆₃N₇O₉S+H, calc:866.4). Compound V was used directly in the next reaction withoutfurther purification.

Preparation of Compound X

To a solution of Compound V (73.3 g, 78.14 mmol, as HCl salt),N-carboxymethylmalonic acid tert-butyl ester (Compound W) (17.0 g, 78.34mmol), and NEt₃ (33.0 mL, 236.7 mmol) in DMF (500 mL) at 0° C. was addedHATU (30.6 g, 80.47 mmol) in portions over 10 min. The reaction mixturewas stirred at ambient temperature for 1 h. Water (500 mL) was added andthe mixture was extracted with EtOAc (750 mL). The EtOAc extracts werewashed with water (2×250 mL), NaHCO₃ (2×200 mL) and brine (250 mL). Theorganic layer was dried over Na₂SO₄ and filtered. The solution wasconcentrated, and the residue was purified by a silica gel column, usinggradient 1-10% MeOH in CH₂Cl₂, to provide Compound X in 43% yield (36.0g, 33.8 mmol) as a white solid. LC-MS [M+H] 1067.2 (C₅₃H₇₆N₈O₁₃S+H,calc: 1065.5).

Preparation ofN-1-[3-(oxycodone-6-enol-carbonyl-methyl-amino)-2,2-dimethyl-propylamine]-arginine-glycine-malonicacid (Compound KC-8)

Compound X (36.0 g, 33.8 mmol) was treated with a mixture of TFA (60 mL)and m-cresol (2.0 mL) at ambient temperature. The reaction progress wasmonitored by LC/MS. After 4 h, the mixture was concentrated in vacuo toremove most of the volatiles (˜90% solvent removed). The residue wastreated with ethyl ether (1 L), and a white precipitate was formed. Theclear supernatant was removed and the precipitate was washed with ethylether (1 L). The solid was then concentrated and subjected to HPLCpurification. [Nanosyn-Pack Microsorb (100-10) C-18 column (50×300 mm);flow rate: 100 mL/min; injection volume 15 mL; mobile phase A: 100%water, 0.1% TFA; mobile phase B: 100% ACN, 0.1% TFA; gradient elutionfrom 0% to 20% B in 30 min, isocratic elution at 20% B in 30 min,gradient elution from 20% B to 45% B in 35 min; detection at 254 nm].Fractions containing the desired compound were combined and concentratedin vacuo. The residue was dissolved in ACN (60 mL) and 0.1 N HCl (200mL), and lyophilized to provide Compound KC-8 in 69.6% yield (19.5 g,23.5 mmol, 99.4% purity) as a white foam. LC-MS [M+H] 758.5(C₃₆H₅₂N₈O₁₀+H, calc: 757.4).

Biological Data Example 4 Pharmacokinetics of Compound KC-8 Following POAdministration to Rats

This Example demonstrates the release of oxycodone into plasma whenCompound KC-8 is administered orally (PO) to rats.

Saline solutions of Compound KC-8 (which can be prepared as described inthe examples herein) were dosed as indicated in Table 1 via oral gavageinto jugular vein-cannulated male Sprague Dawley rats (4 per group) thathad been fasted for 16-18 h prior to oral dosing. At specified timepoints, blood samples were drawn, harvested for plasma viacentrifugation at 5,400 rpm at 4° C. for 5 min, and 100 microliters (μl)plasma transferred from each sample into a fresh tube containing 2 μl of50% formic acid. The tubes were vortexed for 5-10 seconds, immediatelyplaced in dry ice, and then stored in a −80° C. freezer until analysisby HPLC/MS.

Table 1 and FIG. 4 provide oxycodone exposure results for ratsadministered different doses of Compound KC-8. Results in Table 1 arereported, for each group of rats, as (a) maximum plasma concentration(Cmax) of oxycodone (OC) (average±standard deviation), (b) time afteradministration of Compound KC-8 to reach maximum oxycodone concentration(Tmax) (average±standard deviation) and (c) area under the curve (AUC)from 0 to 24 h (average±standard deviation).

TABLE 1 Cmax, Tmax and AUC values of oxycodone in rat plasma Dose, DoseOC Cmax ± sd, AUC ± sd, mg/kg μmol/kg ng/mL Tmax ± sd, h ng * h/mL 2.83.4 0.281 ± 0.49* 2.00 ± 0.0 0.373 ± 0.65 5 6.0  1.39 ± 0.84{circumflexover ( )}  2.00 ± 0.0 5.34 ± 1.9 10 12 3.47 ± 1.6*  2.25 ± 0.50 13.9 ±3.2 23 28 10.4 ± 3.0{circumflex over ( )}   1.75 ± 0.50 41.1 ± 18 45 5414.7 ± 9.3* 2.75 ± 1.5 52.9 ± 24 50 60 21.9 ± 5.3* 2.00 ± 0.0 83.8 ± 24*Lower limit of quantitation was 0.500 ng/mL {circumflex over ( )}Lowerlimit of quantitation was 0.100 ng/mL

FIG. 4 compares mean plasma concentrations over time of oxycodonerelease following PO administration of increasing doses of Compound KC-8to rats.

The results in Table 1 and FIG. 4 indicate that plasma concentrations ofoxycodone increase proportionally with Compound KC-8 dose in rats.

Example 5 Pharmacokinetics of Compound KC-8 Following PO Administrationto Dogs

This Example demonstrates the release of oxycodone into plasma whenCompound KC-8 was administered orally (PO) to dogs. This Example alsocompares such release to that of Compound KC-3, an oxycodone prodrugthat, unlike Compound KC-8, does not contain geminal dimethyl groups inits cyclizable spacer leaving group and lacks glycine in itstrypsin-cleavable moiety. Also compared are oxycodone plasma levels indogs administered oxycodone or OxyContin® tablets.

Study A.

Purebred male young adult/adult beagles were fasted overnight.Increasing doses of Compound KC-8 (as indicated in Table 2A), 4.15 mg/kg(5.7 μmol/kg) of Compound KC-3 (each of which can be prepared asdescribed in the examples herein), or 2 mg/kg (5.7 μmol/kg) oxycodoneHCl (Johnson Matthey Pharmaceutical Materials, West Deptford, N.J., USA)were administered in water via oral gavage (Table 2A indicates thenumber of dogs per group). In addition, one group of 4 dogs wasadministered one 20-mg OxyContin® Tablet (oxycodone HClcontrolled-release) C-II per dog (NDC 59011-420-10, Purdue Pharma,Stamford, Conn., USA). The tablet dose was followed by approximately 5mL of water to facilitate swallowing. The doses of oxycodone and ofOxyContin® tablets were selected to provide approximately equimoleamounts. Blood was collected from each animal via a jugular vein atvarious times over a 24-h period, centrifuged, and 0.8 mL plasmatransferred to a fresh tube containing 8 μL formic acid; samples werevortexed, then immediately placed in dry ice, and stored in a −80° C.freezer until analysis by HPLC/MS.

Table 2A and FIG. 5 provide oxycodone exposure results for dogsadministered the indicated compounds. Results in Table 2A are reported,for each group of dogs, as (a) maximum plasma concentration (Cmax) ofoxycodone (OC) (average±standard deviation), (b) time afteradministration of compound to reach maximum oxycodone concentration(Tmax) (average±standard deviation) and (c) area under the curve (AUC)from 0 to 24 h (average±standard deviation).

TABLE 2A Cmax, Tmax and AUC values of oxycodone in dog plasma Num- Dose,OC AUC ± sd ber Dose, μmol/ Cmax ± Tmax ± sd, (ng × h)/mL of Compoundmg/kg kg sd, ng/mL h 0-24 h dogs KC-8 4.55 5.5 23.5 ± 1.8 1.00 ± 0.0 159 ± 12 3 9.1 11 36.2 ± 4.4 2.33 ± 1.2  277 ± 20 3 KC-3 4.15 5.7 10.2 ±3.3 4.00 ± 0.00 65.6 ± 22  4 Oxycodone 2 5.7 193 ± 69 0.50 ± 0.00 418 ±54 4 Oxy- 20 mg 64.7 ± 8.8 2.75 ± 0.96  329 ± 160 4 Contin ® TabletLower limit of quantitation was 0.0250 ng/mL

FIG. 5 compares mean plasma concentrations over time of oxycodonefollowing PO administration of Compound KC-8, Compound KC-3, OxyContin®Tablets or oxycodone HCl to dogs.

The results in Table 2A and FIG. 5 indicate that oral administration ofCompound KC-8 to dogs leads to a suppressed oxycodone Cmax, delayedoxycodone Tmax and extended oxycodone exposure time (AUC) compared toadministration of oxycodone. Compound KC-8 also provides forsignificantly enhanced release of oxycodone into dog plasma (higher Cmaxand AUC) than does Compound KC-3. The plasma PK profile of oxycodonerelease by Compound KC-8 administered orally to dogs resembles that ofOxyContin® tablets more than that of oxycodone; duration of drugexposure is at least as long for Compound KC-8 as for OxyContin®tablets.

Study B.

Compound KC-8 was also dosed to dogs in a separate experiment at thedoses indicated in Table 2B, and samples were collected at various timesover a 48-h period. Otherwise, the procedures were the same as thatdescribed for Study A.

Table 2B provide oxycodone exposure results for dogs administeredincreasing doses of Compound KC-8. Results are reported as described forTable 2A, except AUC is calculated from 0 to 48 h.

TABLE 2B Cmax, Tmax and AUC values of oxycodone in dog plasma Num- OCAUC ± sd ber Com- Dose, Dose, Cmax ± sd, Tmax ± (ng × h)/mL of poundmg/kg μmol/kg ng/mL sd, h 0-48 h dogs KC-8 4.55 5.48 17.2 ± 9.8** 2.00 ±1.4 123 ± 42 4 KC-8 9.1^(#) 11.0 29.8 ± 9.1§  2.00 ± 0.0 264 ± 71 4 KC-818.2 21.9 63.1 ± 5.4** 3.50 ± 1.7 589 ± 56 4 §Lower limit ofquantitation was 0.100 ng/mL **Lower limit of quantitation was 0.0125ng/mL ^(#)Dosed on a different day

The results in Table 2B show that Compound KC-8 has a reproducible oralPK profile in dogs that is dose proportional.

Example 6 In Vitro Trypsin-Mediated Prodrug Cleavage and Spacer LeavingGroup Cyclization Rate of Compound KC-8

This Example assesses the ability of trypsin to cleave oxycodone prodrugCompound KC-8. This Example also assesses the rate of cyclization andrelease of oxycodone by Compound KC-22, which is identical to CompoundKC-8 except that Compound KC-22 lacks the trypsin-cleavable moiety.

Compound KC-8 was incubated with trypsin from bovine pancreas (CatalogNo. T8003, Type I, ˜10,000 BAEE units/mg protein, Sigma-Aldrich, St.Louis, Mo., USA). Specifically, the reactions included 0.761 mM ofCompound KC-8•2HCl, 22.5 mM calcium chloride, 40 to 172 mM Tris pH 8 and0.25% DMSO with variable activities of trypsin. The reactions wereconducted at 37° C. for 24 h. Samples were collected at specified timepoints, transferred into 0.5% formic acid in acetonitrile to stoptrypsin activity and stored at less than −70° C. until analysis byLC-MS/MS.

Cyclization release rates were measured by following the rate ofdisappearance of Compound KC-22 (2.18 mM initial concentration) in a 50mM pH 7.4 phosphate buffer at 20° C.

Table 3 indicates the results of exposure of Compound KC-8 to trypsin.The results are expressed as half-life of prodrug when exposed totrypsin (i.e., Prodrug trypsin half-life) in hours and rate of oxycodoneformation in μ moles per hour per BAEE unit (μmol/h/BAEE U) trypsin.Table 3 also indicates the cyclization rate of the cyclizable spacerleaving group of Compound KC-22. The results are expressed as half-lifeof compound disappearance.

TABLE 3 In vitro trypsin cleavage of Compound KC-8, and cyclization rateof Compound KC-22 Prodrug trypsin OC formation rate, DisappearanceProdrug half-life, h μmol/h/BAEE U Compound half-life, h KC-8 0.188 ±0.0080 0.296 ± 0.044 KC-22 10.4 ± 0.0060 * Adjusted to 4815 BAEE U/mLtrypsin

The results in Table 3 indicate that Compound KC-8 can be cleaved bytrypsin, and that the spacer leaving group of Compound KC-8 can cyclize,the latter result being shown directly by the cyclization rate ofCompound KC-22.

Example 7 Oral Administration of Compound KC-8 Co-Dosed with TrypsinInhibitor Compound 109 to Rats

This Example demonstrates the ability of a trypsin inhibitor to affectthe ability of Compound KC-8 to release oxycodone into plasma whenCompound KC-8 is administered orally to rats.

Saline solutions of prodrug Compound KC-8 (which can be prepared asdescribed in the examples herein) were dosed as indicated in Table 4.The rats were co-dosed with increasing concentrations of trypsininhibitor Compound 109 (Catalog No. 3081, Tocris Bioscience, Ellisville,Mo., USA, or Catalog No. WS38665, Waterstone Technology, Cannel, 1N,USA) via oral gavage into jugular vein-cannulated male Sprague Dawleyrats (4 per group) that had been fasted for 16-18 h prior to oraldosing. At specified time points, blood samples were drawn, harvestedfor plasma via centrifugation at 5,400 rpm at 4° C. for 5 min, and 100microliters (μl) plasma transferred from each sample into a fresh tubecontaining 2 μl of 50% formic acid. The tubes were vortexed for 5-10seconds, immediately placed in dry ice, and then stored in a −80° C.freezer until analysis by HPLC/MS.

TABLE 4 Co-dosing of prodrug Compound KC-8 and trypsin inhibitorCompound 109 Compound KC-8 Compound KC-8 Compound 109 Compound 109 Dose,mg/kg Dose, μmol/kg Dose, mg/kg Dose, μmol/kg 5 6 0 0 5 6 0.1 0.2 5 60.5 0.9 5 6 1 1.9 50 60 0 0 50 60 1 1.9 50 60 5 9 50 60 10 19

FIG. 6A and FIG. 6B provide oxycodone exposure results for ratsadministered with different doses of Compound KC-8 in the presence orabsence of Compound 109.

FIG. 6A compares mean plasma concentrations over time of oxycodonerelease following PO administration of 5 mg/kg (6 μmol/kg) prodrugCompound KC-8 with increasing amounts of co-dosed trypsin inhibitorCompound 109 to rats.

FIG. 6B compares mean plasma concentrations over time of oxycodonerelease following PO administration of 50 mg/kg (60 μmol/kg) prodrugCompound KC-8 with increasing amounts of co-dosed trypsin inhibitorCompound 109 to rats.

The results in FIG. 6A and FIG. 6B indicate Compound 109's ability toattenuate Compound KC-8's ability to release oxycodone in rats in a dosedependent manner, as indicated by suppressed Cmax and/or delayed Tmax.

Example 8 Oral Administration of a Single Dose Unit and of Multiple DoseUnits of a Composition Comprising Prodrug Compound KC-8 and TrypsinInhibitor Compound 109 in Rats

This Example demonstrates the effect of oral administration of singleand multiple dose units comprising prodrug Compound KC-8 and trypsininhibitor Compound 109 to rats.

Saline solutions of Compound KC-8 (which can be prepared as described inthe examples herein) were dosed orally to rats (4 rats per group) atincreasing concentrations ranging from 5 to 50 mg/kg (from 6 to 60μmol/kg), wherein a single dose was represented as 5 mg/kg (6 μmol/kg)Compound KC-8 in the absence of trypsin inhibitor.

A second set of rats (4 rats per group) were co-dosed orally withprodrug Compound KC-8 and trypsin inhibitor Compound 109 (Catalog No.3081, Tocris Bioscience, or Catalog No. WS38665, Waterstone Technology)as described below and indicated in Table 5. Specifically, a salinesolution of a composition comprising 5 mg/kg (6 μmol/kg) Compound KC-8and 0.5 mg/kg (1 μmol/kg) Compound 109, representative of a single doseunit, was administered via oral gavage to a group of 4 rats. It is to benoted that the mole-to-mole ratio of trypsin inhibitor-to-prodrug(109-to-KC-8) is 0.17-to-1; as such this dose unit is referred to hereinas a 109-to-KC-8 (0.17-to-1) dose unit. Saline solutions representativeof 2 dose units, 3 dose units, 4 dose units, 6 dose units, 8 dose units,and 10 dose units (i.e., as indicated in Table 5) of the 109-to-KC-8(0.17- to 1) dose unit were similarly administered to additional groupsof 4 rats.

All rats were jugular vein-cannulated male Sprague Dawley rats that hadbeen fasted for 16-18 h prior to oral dosing. Dosing, sampling andanalysis procedures were similar to those described in Example 4.

Table 5 (top half) and FIG. 7A provide oxycodone exposure results inplasma for rats administered 1, 2, 3, 4, 6, 8 and 10 doses of CompoundKC-8 in the absence of trypsin inhibitor. Table 5 (bottom half) and FIG.7B provide oxycodone exposure results in plasma for rats administered 1,2, 3, 4, 6, 8 and 10 dose units of the 109-to-KC-8 (0.17-to-1) doseunit. Oxycodone Cmax, Tmax and AUC values are reported as described inExample 4.

TABLE 5 Cmax, Tmax and AUC values of oxycodone in rat plasma KC-8 KC-8109 109 OC Cmax ± Amount Dose, Dose, Dose, Dose, sd, Tmax ± AUC ± sd,(Multiple) mg/kg μmol/kg mg/kg μmol/kg ng/mL sd, h ng * h/mL  1 KC-8dose 5 6 0 0* 1.39 ± 0.84 2.00 ± 0.0  5.34 ± 1.9  2 KC-8 doses 10 12 00^(#) 3.47 ± 1.6  2.25 ± 0.50 13.9 ± 3.2  3 KC-8 doses 15 18 00{circumflex over ( )} 4.23 ± 2.2  2.50 ± 0.58 24.3 ± 16   4 KC-8 doses20 24 0 0{circumflex over ( )} 3.68 ± 1.7  2.25 ± 0.50 25.2 ± 14   6KC-8 doses 30 36 0 0{circumflex over ( )} 7.42 ± 1.7  3.50 ± 3.0  65.1 ±25   8 KC-8 doses 40 48 0 0{circumflex over ( )} 9.16 ± 5.3  2.25 ± 0.5045.1 ± 26  10 KC-8 doses 50 60 0 0^(#) 21.9 ± 5.3  2.00 ± 0.0  83.8 ±24   1 dose unit 5 6 0.5 1* 1.09 ± 0.55 3.25 ± 1.3  5.66 ± 2.0  2 doseunits 10 12 1 2{circumflex over ( )} 2.82 ± 0.97 3.50± 1.0  11.6 ± 1.5 3 dose units 15 18 1.5 3{circumflex over ( )} 2.13 ± 0.75 4.50 ± 1.0 10.3 ± 4.3  4 dose units 20 24 2 4{circumflex over ( )} 3.34 ± 2.1  7.25± 1.5  14.6 ± 9.1  6 dose units 30 36 3 6{circumflex over ( )} 3.27 ±1.2  5.00 ± 0.0  28.6 ± 19   8 dose units 40 48 4 7{circumflex over ( )}4.63 ± 3.3  4.50 ± 1.0  45.2 ± 39  10 dose units 50 60 5 9^(#) 4.24 ±0.71 6.50 ± 1.7  20.0 ± 3.5 *Lower limit of quantitation was 0.100 ng/mL{circumflex over ( )}Lower limit of quantitation was 0.0500 ng/mL^(#)Lower limit of quantitation was 0.500 ng/mL

FIG. 7A compares mean plasma concentrations over time of oxycodonerelease following PO administration of a single dose and of multipledoses of Compound KC-8 dosed in the absence of trypsin inhibitor.

FIG. 7B compares mean plasma concentrations over time of oxycodonerelease following PO administration of a single dose unit and ofmultiple dose units of a composition comprising prodrug Compound KC-8and trypsin inhibitor Compound 109.

The results in Table 5, FIG. 7A and FIG. 7B indicate that administrationof multiple dose units (as exemplified by 1, 2, 3, 4, 6, 8 and 10 doseunits of the 109-to-KC-8 (0.17- to 1) dose unit) results in a plasmaoxycodone concentration-time PK profile that is not dose proportional tothe plasma oxycodone concentration-time PK profile of the single doseunit. In addition, the PK profile of the multiple dose units (e.g., FIG.7B) was modified compared to the PK profile of the equivalent dosage ofprodrug in the absence of trypsin inhibitor (e.g., FIG. 7A).

Example 9 Oral Administration of Compound KC-8 Co-Dosed with TrypsinInhibitor Compound 109 to Dogs

This Example demonstrates the ability of a trypsin inhibitor to affectthe ability of Compound KC-8 to release oxycodone into plasma whenCompound KC-8 is administered orally (PO) to dogs.

Purebred male young adult/adult beagles were fasted overnight. CompoundKC-8 (which can be prepared as described in the examples herein) wasadministered at 18.2 mg/kg (22 μmol/kg) with or without a co-dose of 1.8mg/kg (3.3 μmol/kg) Compound 109 (Catalog No. 3081, Tocris Bioscience,or Catalog No. WS38665, Waterstone Technology) in water via oral gavageas indicated in Table 6. Blood was collected, treated and analyzed as inExample 5.

Table 6 and FIG. 8 provide oxycodone exposure results for dogsadministered Compound KC-8, in the presence or absence of Compound 109.Results in Table 6 are reported for each group of four dogs, asdescribed in Example 5.

TABLE 6 Dog dosing PO with Compound KC-8 in the absence or presence ofCompound 109 KC-8 KC-8 Dose, Compound Compound OC Tmax ± AUC ± Dose,μmol/ 109 Dose, 109 Dose, Cmax ± sd, sd, mg/kg kg mg/kg μmol/kg sd,ng/mL h ng * h/mL 18.2 22 0 0 63.1 ± 5.4 3.50 ± 1.7  589 ± 56 18.2 221.8 3.3 8.86 ± 1.8 8.00 ± 0.0 98.4 ± 25 Lower limit of quantitation was0.0125 ng/mL

FIG. 8 compares mean plasma concentrations over time of oxycodonerelease following PO administration to dogs of Compound KC-8 with orwithout a co-dose of trypsin inhibitor Compound 109.

The results in Table 6 and FIG. 8 indicate Compound 109's ability toattenuate Compound KC-8's ability to release oxycodone, both bysuppressing Cmax and AUC and by delaying Tmax.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A composition comprising a trypsin inhibitor andN-1-[3-(oxycodone-6-enol-carbonyl-methyl-amino)-2,2-dimethyl-propylamine]-arginine-glycine-malonicacid, Compound KC-8, shown below:

or acceptable salts thereof.
 2. The composition of claim 1, wherein thetrypsin inhibitor is an arginine mimic or a lysine mimic.
 3. Thecomposition of claim 2, wherein the arginine mimic or lysine mimic is asynthetic compound.
 4. The composition of claim 1, wherein the trypsininhibitor is a compound of formula:

wherein: Q¹ is selected from —O-Q⁴ or -Q⁴-COOH, where Q⁴ is C₁-C₄ alkyl;Q² is N or CH; and Q³ is aryl or substituted aryl.
 5. The composition ofclaim 1, wherein the trypsin inhibitor is a compound of formula:

wherein: Q⁵ is —C(O)—COOH or —NH-Q⁶-Q⁷-SO₂—C₆H₅, where Q⁶ is—(CH₂)_(p)—COOH; Q⁷ is —(CH₂)_(r)—C₆H₅; Q⁸ is NH; n is a number fromzero to two; o is zero or one; p is an integer from one to three; and ris an integer from one to three.
 6. The composition of claim 1, whereinthe trypsin inhibitor is a compound of formula:

wherein: Q⁵ is —C(O)—COOH or —NH-Q⁶-Q⁷-SO₂—C₆H₅, where Q⁶ is—(CH₂)_(p)—COOH; Q⁷ is —(CH₂)_(r)—C₆H₅; and p is an integer from one tothree; and r is an integer from one to three.
 7. The composition ofclaim 1, wherein the trypsin inhibitor is a compound of formula:

wherein X is NH; n is zero or one; and R^(t1) is selected from hydrogen,halogen, nitro, alkyl, substituted alkyl, alkoxy, carboxyl,alkoxycarbonyl, acyl, aminoacyl, guanidine, amidino, carbamide, amino,substituted amino, hydroxyl, cyano and—(CH₂)_(m)—C(O)—O—(CH₂)_(m)—C(O)—N—R^(n1)R^(n2), wherein each m isindependently zero to 2; and R^(n1) and R^(n2) are independentlyselected from hydrogen and C₁₋₄ alkyl.
 8. The composition of claim 1,wherein the trypsin inhibitor is a compound of formula:

wherein X is NH; n is zero or one; L^(t1) is selected from —C(O)—O—;—O—C(O)—; —O—(CH₂)_(m)—O—; —OCH₂—Ar^(t2)-CH₂O—; —C(O)—NR^(t3)—; and—NR^(t3)—C(O)—; R^(t3) is selected from hydrogen, C₁₋₆ alkyl, andsubstituted C₁₋₆ alkyl; Ar^(t1) and Ar^(t2) are independently asubstituted or unsubstituted aryl group; m is a number from 1 to 3; andR^(t2) is selected from hydrogen, halogen, nitro, alkyl, substitutedalkyl, alkoxy, carboxyl, alkoxycarbonyl, acyl, aminoacyl, guanidine,amidino, carbamide, amino, substituted amino, hydroxyl, cyano and—(CH₂)_(m)—C(O)—O—(CH₂)_(m)—C(O)—N—R^(n1)R^(n2), wherein each m isindependently zero to 2; and R^(n1) and R^(n2) are independentlyselected from hydrogen and C₁₋₄ alkyl.
 9. The composition of claim 1,wherein the trypsin inhibitor is a compound of formula:

wherein each X is NH; each n is independently zero or one; L^(t1) isselected from —C(O)—O—; —O—C(O)—; —O—(CH₂)_(m)—O—; —OCH₂—Ar^(t2)-CH₂O—;—C(O)—NR^(t3)—; and —NR^(t3)—C(O)—; R^(t3) is selected from hydrogen,C₁₋₆ alkyl, and substituted C₁₋₆ alkyl; Ar^(t1) and Ar^(t2) areindependently a substituted or unsubstituted aryl group; and m is anumber from 1 to
 3. 10. The composition of claim 1, wherein the trypsininhibitor is selected from (S)-ethyl4-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperazine-1-carboxylate;(S)-ethyl4-(5-guanidino-2-(2,4,6-triisopropylphenylsulfonamido)pentanoyl)piperazine-1-carboxylate;(S)-ethyl1-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperidine-4-carboxylate;(S)-ethyl1-(5-guanidino-2-(2,4,6-triisopropylphenylsulfonamido)pentanoyl)piperidine-4-carboxylate;(S)-6-(4-(5-guanidino-2-(naphthalene-2-sulfonamido)pentanoyl)piperazin-1-yl)-6-oxohexanoicacid; 4-aminobenzimidamide; 3-(4-carbamimidoylphenyl)-2-oxopropanoicacid;(S)-5-(4-carbamimidoylbenzylamino)-5-oxo-4-((R)-4-phenyl-2-(phenylmethylsulfonamido)butanamido)pentanoicacid; 6-carbamimidoylnaphthalen-2-yl-4-(diaminomethyleneamino)benzoate;and 4,4′-(pentane-1,5-diylbis(oxy))dibenzimidamide.
 11. The compositionof claim 1, wherein the trypsin inhibitor is6-carbamimidoylnaphthalen-2-yl-4-(diaminomethyleneamino)benzoate(Compound 109).
 12. A method of treating or preventing pain in a patientin need thereof, which comprises administering an effective amount of acomposition of claim 1 to the patient.